JP2021515576A - B cells genetically engineered to secrete follistatin and methods of using it to treat follistatin-related diseases, conditions, disorders, and to enhance muscle growth and strength. - Google Patents

Abstract

The present invention relates to methods for administering autologous and / or allogeneic B cells genetically modified to produce therapeutic agents such as follistatin. Specific disclosures include a method for administering a single maximum effective dose of genetically modified B cells, and a method for administering multiple doses of follistatin-expressing genetically modified B cells. The compositions and methods disclosed herein are useful for long-term in vivo delivery of follistatin.

Description

Cross-reference to related applications This application is filed on March 16, 2018, US Provisional Application No. 62 / 644,362, and on March 16, 2018, US Provisional Application No. 62 / 644. Claim priority to No. 356, each of which is incorporated herein by reference in its entirety.

Sequence Listing Statements The sequence listings related to this application are provided in text format instead of a paper copy and are incorporated herein by reference. The name of the text file containing the sequence listing is IMCO-008_01WO_ST25. It is txt. This text file, 12KB, was created on March 18, 2019 and submitted electronically via EFS-Web.

Background The present disclosure relates to the use of B cells for long-term in vivo delivery of therapeutic agents, eg, follistatin, in particular to single and multiple doses of B cells to a subject (eg, human). Regarding administration.

Description of Related Techniques Muscular dystrophy (MD) is a progressive hereditary neuromuscular disorder characterized by muscle wasting and weakness (Emery (2002) The Lancet, 359: 687-695). Many forms of muscular dystrophy are lethal and are currently untreatable.

Duchenne muscular dystrophy (DMD) is the most common X-linked neuromuscular disease. The disease is caused by mutations in the DMD gene that encodes dystrophin. Modification or absence of this protein results in abnormal sarcolemma rupture. Abnormal variation in diameter of muscle fibers (atrophic and hypertrophic fibers) in the proximal muscle and progressive muscle damage are characteristic of the disease. Damaged muscle releases the intracellular enzyme creatine kinase (CK). As a result, serum CK levels in DMD patients are characterized by high levels (up to 10 times normal values). The pathophysiological cascade consists of tissue inflammation, muscle fiber necrosis and muscle replacement by fibrous adipose tissue.

Another allelic variant of the DMD gene causes a milder form of MD known as Becker muscular dystrophy (BMD). BMD is clinically similar to DMD, but the onset of symptoms occurs in later years.

Many drugs have been tried in MD, but none have proven effective in controlling the course of the disease. Current treatment modality is still within the scope of physical therapy and rehabilitation.

Several clinical trials using corticosteroids (eg, prednisone and / or its derivatives) have demonstrated improvement in individuals with MD, especially in the short term. Although the exact mechanism by which corticosteroids alleviate the phenotype of the disease is unclear, corticosteroids reduce inflammation, suppress the immune system, improve calcium homeostasis, and compensatory proteins. It is thought to act by upregulating the expression of and increasing the proliferation of myoblasts (Khurana et al. (2003) Nat. Rev. Drug Discovery 2: 279-386). However, corticosteroids administered over time induce muscle atrophy, which can primarily affect the proximal muscles, which are exactly the same muscles affected in DMD and BMD. Muscle-induced effects and other side effects of corticosteroids may limit the long-term efficacy of corticosteroid therapy.

The transforming growth factor-beta (TGF-beta) superfamily contains a variety of growth factors that share common sequence elements and structural motifs. These proteins are known to exert biological effects on a variety of cell types in both vertebrates and non-vertebrates. Members of this superfamily play important roles during embryogenesis in patterning and tissue differentiation, including fat production, muscle development, chondrogenesis, cardiac development, hematopoiesis, neurogenesis, and epithelial cell differentiation. Can affect the differentiation process. The family is divided into two common branches: BMP / GDF and TGF-beta / activin / BMP10, and these members have various, often complementary effects. Manipulating the activity of members of the TGF-beta family can often result in significant physiological changes in the organism. For example, bovine breeds of Piedmontese and Belgian Blue carry a loss-of-function mutation in the GDF8 (also known as myostatin) gene that causes a marked increase in muscle mass. Grobet et al., Nat. Genet. 1997, 17 (1): 71-4. Moreover, in humans, the GDF8 inactive allele is associated with increased muscle mass and, reportedly, exceptional intensity. Schuelke et al., N Engl J Med 2004, 350: 2682-8. In addition, mice genetically engineered to express the dominant negative activin receptor IIB (ActRIIB) or follistatin have exceptional muscle mass (Lee, SJ and McPherron, AC, Proc Natl Acad Sci). US A. 2001 Jul 31; 98 (16): 9306-11), Overexpression of follistatin in non-human primates enhances muscle growth and strength. Kota J, et al., Sci Transl Med. 2009 Nov 11; 1 (6).

Therefore, there is a need for a method of delivering a drug that functions as an effective regulator of TGF-beta signaling.
Current methods for treating chronic diseases and disorders include direct infusion of therapeutic agents (eg, therapeutic polypeptides), gene therapy with viral vectors, and stem cell adoption (eg, hematopoietic stem cell transfer). However, each of these methods has its disadvantages. Injection of recombinant therapeutic proteins suffers from a finite half-life of the protein, and all three methods result in suboptimal tissue penetration with therapeutic agents. Modifying the endogenous tissue to produce a therapeutic agent, such as by injection of recombinant adeno-associated virus (AAV) and lentiviral vectors, generally results in a therapeutic agent produced from a centrally concentrated location. Production of the therapeutic agent from one location increases the probability of localized toxicity in the producing tissue. Moreover, because recombinant viruses are considered foreign, it is unlikely that the viral vector can be administered multiple times without causing adverse reactions, simply to achieve an accurate dose of therapeutic agent. It means that there is an opportunity for multiple injections. Given the biological variations inherent in procedures such as in vivo introduction of nucleic acids into cells using the virus, it would be highly unacceptable to achieve the desired dose under the constraints of a single injection.

Emery (2002) The Lancet, 359: 687-695 Khurana et al. (2003) Nat. Rev. Drug Discovery 2: 279-386 Grobet et al., Nat. Genet. 1997, 17 (1): 71-4 Schuelke et al., N Engl J Med 2004, 350: 2682-8 Lee, SJ and McPherron, AC, Proc Natl Acad Sci U S A. 2001 Jul 31; 98 (16): 9306-11 Kota J, et al., Sci Transl Med. 2009 Nov 11; 1 (6)

Therefore, there is still a need for long-term treatment in the art for many chronic diseases and disorders associated with TGF-beta signaling.

Summary of Embodiments The present disclosure generally relates to compositions and methods for administering and administering genetically modified B cell compositions to treat chronic diseases and disorders. In various embodiments, the present disclosure is for administering and administering B cells genetically modified to express a polypeptide capable of modulating TGF-beta signaling (eg, a follistatin polypeptide). Compositions and methods of. In some specific embodiments, the present disclosure relates to compositions and methods for administering and administering B cells genetically modified to express a follistatin polypeptide. In certain embodiments, the present disclosure administers and administers B cells genetically modified to express a follistatin polypeptide having the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. Concerning compositions and methods for dosing. Such B cells can be used in various embodiments, for example, to increase muscle size or strength in a subject (eg, human). The present disclosure provides the above and other advantages as described in the detailed description.

In some embodiments, the invention provides recombinant B cells containing the follistatin gene. In some embodiments, the follistatin gene is operably linked to a promoter. In some embodiments, the follistatin gene is a human follistatin gene. In some embodiments, the follistatin gene is a splice site variant of human follistatin FST-344. In some embodiments, the B cells are human B cells. In some embodiments, B cells are transduced with the follistatin gene. In some embodiments, B cells contain the follistatin gene because they have been transduced with the follistatin gene using a sleeping beauty transposon system. In some embodiments, B cells express the follistatin gene by transduction with a virus carrying the follistatin gene. In some embodiments, the B cell contains the follistatin gene because it has been transduced by a retrovirus containing the follistatin gene, a lentivirus, an adenovirus or an adeno-associated virus. In some embodiments, B cells are engineered to contain the follistatin gene using a targeted integration technique. In some embodiments, targeted integration includes, but is not limited to, one or more zinc finger nucleases, transcriptional activator-like effector nucleases (TALENs), and / or CRISPR / Cas9 systems. Use the system. In some embodiments, B cells are selected from the group consisting of retroviral vectors, lentiviral vectors, adeno-associated virus vectors, adenoviral vectors, any other RNA or DNA viral vectors. By introducing a nucleic acid encoding a follistatin, the follistatin gene is introduced using such and chemical or physical means such as lipofection, polycation complex formation, electroporation, etc. It is engineered to contain non-viral DNA and / or RNA encoding statin. In some embodiments, the follistatin gene is secreted by recombinant B cells. In some embodiments, the recombinant B cells are derived from B cells obtained from a subject or B cells derived from cells obtained from a subject. In some embodiments, the recombinant B cells are derived from B cell progenitor cells obtained from the subject. In some embodiments, recombinant B cells are derived from cells obtained from a subject that have differentiated into B cells or progenitor cells of B cells. In some embodiments, recombinant B cells are
(A) With the steps of collecting and isolating immune cells from the subject's blood;
(B) The step of transducing a DNA encoding follistatin into a cell;
(C) With the step of expanding and proliferating the selected cells ex vivo;
(D) Manipulated by steps to differentiate ex vivo expanded and proliferated cells into plasma cells and / or plasma blast cells.

In some embodiments, the immune cells isolated from step a are CD19 positive cells. In some embodiments, the transduction step of step b is by electroporation. In some embodiments, a sleeping beauty transposon system was utilized for electroporation. In some embodiments, the differentiated cells are CD38 (+) and CD20 (−). In some embodiments, the invention provides a method comprising the step of administering such recombinant B cells to a subject.

In some embodiments, the invention provides a method of delivering follistatin to a subject in need thereof, comprising the step of administering recombinant B cells containing the follistatin gene. In some embodiments, the invention is a method of delivering follistatin to a subject in need thereof, the recombinant B cells disclosed herein expressing the follistatin polypeptide. Provided is a method comprising the step of administering any one of the above to a subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject has a muscular disorder. In some embodiments, the muscular disorder is muscular dystrophy. In some embodiments, the muscular dystrophy is selected from Duchenne muscular dystrophy, Becker muscular dystrophy and fascioscapulohumeral muscular dystrophy. In some embodiments, the myopathy is an inflammatory myopathy. In some embodiments, the inflammatory myopathy is inclusion body myositis. In some embodiments, the muscle disorder is muscle injury or trauma. In some embodiments, the muscle disorder is muscle disuse. In some embodiments, muscle disuse occurs after prolonged bed rest or limb fixation. In some embodiments, the myopathy is muscle atrophy or weakness. In some embodiments, muscle atrophy or weakness is caused by aging, cancer, or chronic disease. In some embodiments, muscle atrophy or weakness is due to muscle weakness. In some embodiments, muscle atrophy or weakness is due to spinal muscular atrophy (SMA). In some embodiments, muscle atrophy or weakness is due to amyotrophic lateral sclerosis (ALS). In some embodiments, muscle atrophy or weakness is due to Pompe disease. In some embodiments, the subject has healthy muscles. In some embodiments, administration of B cells expressing the follistatin polypeptide to a subject having healthy muscle increases the size or strength of the subject's muscles.

In some specific embodiments, the disclosure provides a method for treating muscular dystrophy, comprising the step of administering a follistatin polypeptide-expressing B cell to a subject having muscular dystrophy. .. In some embodiments, the subject has Becker-type muscular dystrophy. In some embodiments, the step of administering recombinant B cells to a subject results in treatment of the subject's disease, disorder, or condition. In some embodiments, the step of administering recombinant B cells to a subject results in treatment of muscular dystrophy. In some embodiments, the step of administering recombinant B cells to a subject increases the body weight of the subject. In some embodiments, the subject gains at least about 4% body weight. In some embodiments, significant weight gain occurs within 30 days. In some embodiments, significant weight gain occurs in about 30 days. In some embodiments, the step of administering recombinant B cells to a subject increases the subject's muscles. In some embodiments, the step of administering recombinant B cells to a subject increases the strength of the subject. In some embodiments, the step of administering recombinant B cells to a subject raises the plasma levels of the subject's follistatin.

In some embodiments, the invention provides a method of treating, preventing, or alleviating muscular dystrophy by administering recombinant B cells containing the follistatin gene. In some embodiments, the method of treating, preventing, or alleviating muscular dystrophy comprises administering any one of the recombinant B cells disclosed herein. In some embodiments, the method comprises administering to the subject two or more sequential doses of genetically modified B cells. In some embodiments, the step of administration comprises two or more doses of genetically modified B cells at a single dose concentration below optimal. In some embodiments, the step of administration comprises a dose of 3 or higher doses of genetically modified B cells. In some embodiments, the genetically modified B cells are autologous to the subject. In some embodiments, the genetically modified B cells are allogeneic to the subject. In some embodiments, the subject is a human. In some embodiments, the genetically modified B cells are CD20-, CD38- and CD138-. In some embodiments, the genetically modified B cells are CD20-, CD38 + and CD138 +. In some embodiments, the genetically modified B cells are CD20-, CD38 + and CD138-. In some embodiments, the step of administration comprises intravenous, intraperitoneal, subcutaneous, or intramuscular injection. In some embodiments, the step of administration comprises an intravenous injection. In some embodiments, the genetically modified B cells are engineered 2 or 3 days after culturing. In some embodiments, genetically modified B cells are engineered using methods involving electroporation. In some embodiments, the genetically modified B cells are collected for administration to the subject on days 4, 5, 6, or 7 of the post-manipulation culture. In some embodiments, genetically modified B cells are collected for administration to a subject on or after day 8 in post-manipulated culture. In some embodiments, genetically modified B cells are collected for administration to a subject on or earlier than day 10 in post-manipulated culture. In some embodiments, the collected genetically modified B cells do not produce significant levels of inflammatory cytokines. In some embodiments, genetically modified B cells are collected at a time in culture where it is determined not to produce significant levels of inflammatory cytokines. In some embodiments, genetically modified B cells are subjected to pre- and post-manipulation culture periods of IL-2, IL-4, IL-10, IL-15, IL-31 and multimerized CD40 ligands. Grow in a culture system containing each. In some embodiments, the multimerized CD40 ligand is a HIS-tagged CD40 ligand that is multimerized using an anti-his antibody. In some embodiments, the method further comprises the step of expanding and proliferating genetically modified B cells prior to the step of administration to the subject. In some embodiments, the final population of expanded and proliferated genetically modified B cells exhibits a high degree of clonality. In some embodiments, any particular B cell clone in the final population of expanded and proliferated genetically modified B cells constitutes less than 0.2% of the total B cell population. In some embodiments, any particular B cell clone in the final population of expanded and proliferated genetically modified B cells constitutes less than 0.05% of the total B cell population. In some embodiments, the genetically modified B cell comprises a polynucleotide encoding the human DHFR gene with enhanced resistance to methotrexate. In some embodiments, the human DHFR gene with enhanced resistance to methotrexate contains a leucine-to-tyrosine substitution mutation at amino acid 22 and a phenylalanine-to-serine substitution mutation at amino acid 31. In some embodiments, the method comprises treating genetically modified B cells with methotrexate prior to collection for administration. In some embodiments, treatment with methotrexate is between 100 nM and 300 nM. In some embodiments, treatment with methotrexate is 200 nM. In some embodiments, genetically modified B cells migrate to diverse tissues when administered to a subject. In some embodiments of the method, at least one of the population of genetically modified B cells administered to a subject is bone marrow, intestine, muscle, spleen, kidney, heart, liver, lung and It migrates to one or more tissues selected from the group consisting of the brain. In some embodiments of the method, at least one of the population of genetically modified B cells administered to a subject has the subject's bone marrow, intestines, muscles, spleen, kidneys, heart, liver. Nominates to the lungs and brain.

In some embodiments, the invention provides modified B cells transduced to express both the follistatin gene and the dihydrofolate reductase (DHFR) gene.

FIG. 1 shows that treatment with B cells expressing follistatin results in increased levels of follistatin in mouse plasma. From left to right in each of the control and treatment groups: Bars correspond to days 21, 28, and 35, respectively.

FIG. 2 shows that plasma levels of follistatin in mice treated with B cells expressing follistatin correlate with levels of human IgG, a surrogate marker for engraftment. 2A-2D show plasma levels of follistatin in four separate mice treated with B cells expressing follistatin. FIG. 2 shows that plasma levels of follistatin in mice treated with B cells expressing follistatin correlate with levels of human IgG, a surrogate marker for engraftment. 2A-2D show plasma levels of follistatin in four separate mice treated with B cells expressing follistatin.

FIG. 3 shows the percentage change in body weight of mice treated or untreated with B cells expressing follistatin.

FIG. 4 shows an assessment of the intensity of mice treated or untreated with B cells expressing follistatin. FIG. 4A shows the strength evaluated by the forelimb grip test. FIG. 4B shows the strength evaluated by the limb grip test. FIG. 4C shows the strength evaluated by the hanging test. The percentage of improvement given below the graph indicates the average percentage of improvement in the treated group compared to the untreated group. FIG. 4 shows an assessment of the intensity of mice treated or untreated with B cells expressing follistatin. FIG. 4A shows the strength evaluated by the forelimb grip test. FIG. 4B shows the strength evaluated by the limb grip test. FIG. 4C shows the strength evaluated by the hanging test. The percentage of improvement given below the graph indicates the average percentage of improvement in the treated group compared to the untreated group.

FIG. 5 shows follistatin expression in vitro in B cells expressing follistatin. FIG. 5A shows the expression of the follistatin protein as determined by ELISA. FIG. 5B shows the expression of follistatin mRNA as determined by RT-PCR.

Detailed Description The practice of the present invention is within the skill of one of ordinary skill in the art, unless otherwise indicated, the conventional practice of molecular biology, recombinant DNA techniques, protein expression and protein / peptide / carbohydrate chemistry. Methods will be used, many of which will be described below for explanatory purposes. Such techniques are well described in the literature. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd edition, 2000); DNA Cloning: A Practical Cloning, Vol. Synthesis: Methods and Applications (edited by P. Herdewijn, 2004); Nucleic Acid Hybridization (edited by B. Hames and S. Higgins, ed., 1985); Cloning (B. Hames and S. Hybrids, 1984); Animal Cell Culture (R. Freshney, 1986); Freshney, R. et al. I. (2005) Culture of Animal Cells, a Manual of Basic Technology, 5th Edition, Hoboken NJ, John Wiley &Sons; B.M. Perbal, A Practical Guide to Molecular Cloning (3rd edition, 2010); Farrell, R. et al. , RNA Methodologies: A Laboratory Guide for Isolation and Comparison (3rd edition, 2005). The publications described above are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an endorsement that the invention does not have the right to precede such disclosure because of the prior invention.

Definitions and Abbreviations Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. As used herein and in the appended claims, the following terms have the meanings shown, unless the opposite is specified. With respect to this specification, whenever the definition of a term defined herein differs from the definition given to this same term in the incorporated references, it is clearly defined herein. The definition is the exact definition of the term.

The words "one (a)" and "one (an)" display one or more unless otherwise noted.

"Approximately" means 30, 25, 20, 15, 10, 9, 8, relative to reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight or length. It means a quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight or length that varies by 7, 6, 5, 4, 3, 2 or 1%. It is specifically contemplated that the term about may be omitted in any embodiment described in the numerical context used in conjunction with the term "about".

The "composition" can include an active agent and carrier, an inert or active carrier, eg, a pharmaceutically acceptable carrier, diluent or excipient. In certain embodiments, the composition is sterile, endotoxin-free, or non-toxic to the recipient at the dosage or concentration used.

Unless the context requires otherwise, throughout the specification and claims, the words "comprise", as well as "comprises" and "comprising", etc. The variant should be interpreted in an open and inclusive sense, i.e., "is, but is not limited to,".

"Consists" means "including and limited to" whatever comes before the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are necessary or essential and that no other element can exist. By "being essentially from" is meant to include any of the elements listed prior to this phrase, which also contributes to the action or action specified in this disclosure for the listed elements. Limited to other factors that may not exist. Thus, the phrase "becomes essential from" means that the enumerated elements are required or compulsory, while the other elements are as needed and the functions of the enumerated elements or It is shown that it may or may not be present depending on whether or not it affects the action.

References of "biological activity" or "biological activity" throughout the specification are in, as a result of administration of any of the compounds, agents, polypeptides, conjugates, pharmaceutical compositions contemplated herein. Refers to any response induced in an in vitro assay or in a cell, tissue, organ or organism (eg, animal or mammal or human). Biological activity can refer to agonistic or antagonistic effects. Biological activity can be a beneficial effect; or biological activity can be non-beneficial, i.e. toxic. In some embodiments, biological activity will refer to the positive or negative effects of a drug or pharmaceutical composition on a living subject, eg, a mammal such as a human. Thus, the term "biologically active" is meant to describe any compound that possesses biological activity, as described herein. Biological activity can be assessed by any suitable means currently known to those of skill in the art. Such assays can be qualitative or quantitative. Those skilled in the art will readily recognize the need to use different assays to assess the activity of different polypeptides; a customary task for the average researcher. Such assays are often easily performed in laboratory settings with little optimization required, and often use a wide range of polypeptides common to many laboratories. Commercial kits are available that provide simple, reliable, and reproducible readings of the biological activity of. When such kits are not available, routinely skilled researchers can easily design and optimize tissue bioactivity assays for target polypeptides without undue experimental design. The reason is that this is a customary aspect of the scientific process.

References to the term "eg" are intended to mean "eg, but not limited to:" and are thus merely examples of a particular embodiment, whatever follows. It should be understood, but it should never be interpreted as a limited example. Unless otherwise noted, the use of "eg" is intended to articulate that other embodiments are contemplated and are incorporated by the present invention.

References throughout the specification of an "embodiment" or "one embodiment" or "an embodiment" or "some embodiments" or "a particular embodiment" are described in connection with an embodiment. It means that a particular feature, structure or feature is included in at least one embodiment of the present invention. Thus, the appearance of the phrase "in one embodiment" or "in one embodiment" or "in a particular embodiment" in various places throughout the specification does not necessarily refer to the same embodiment. In addition, certain features, structures or features can be combined in any suitable manner in one or more embodiments.

An "increased" or "enhanced" amount is typically a "statistically significant" amount and is the amount or level described herein 1.1, 1.2, 1. 3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40 or 50 times or more (eg 100, 500, 1000 times) (any integer and decimal point greater than 1 in between, eg 2.1, 2. It can include an increase (including 2, 2.3, 2.4, etc.). Similarly, a "reduced" or "reduced" or "less" amount is typically a "statistically significant" amount, approximately one of the amounts or levels described herein. .1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4 .5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40 or 1/50 or less (eg, 100, 500, 1/1000) (greater than 1 in between) Any integer and decimal point can be included (including, for example, 1.5, 1.6, 1.7, 1.8, etc.).

The terms "in vitro", "ex vivo" and "in vivo" are intended herein to have their usual scientific significance. Thus, for example, "in vitro" occurs with isolated cell components, such as an enzymatic reaction performed in vitro using appropriate substrates, enzymes, donors and optionally buffers / cofactors. Means to refer to an experiment or reaction. “Ex vivo” means an experiment or reaction performed using a functional organ or cell that has been removed from or propagated independently of the organism. "In vivo" means an experiment or reaction that occurs in a living organism in its normal and intact state.

"Mammals" are both humans and domestic animals such as laboratory animals and domestic pets (eg, cats, dogs, pigs, cows, sheep, goats, horses and rabbits), as well as non-domestic animals such as wildlife and others. And include.

"As needed" or "as needed" means that the event or situation described thereafter may or may not occur, and that the description is when the event or situation occurs. It means that it includes the case where it does not occur.

"Pharmaceutical composition" refers to a compound (eg, a therapeutically useful polypeptide) and a formulation of a medium generally accepted in the art for delivery of a compound to an animal, eg, a human. Such vehicles can therefore include any pharmaceutically acceptable carrier, diluent or excipient.

"Pharmaceutically effective excipients" and "pharmaceutically effective carriers" are well known to those of skill in the art, and methods for their preparation are also readily apparent to those of skill in the art. Such compositions and methods for their preparation can be found, for example, in Remington's Pharmaceutical Sciences, 19th Edition (Mac Publishing Company, 1995, incorporated herein).

The terms "polynucleotide", "nucleotide", "nucleotide sequence" and "nucleic acid" are used interchangeably. These refer to the polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have either three-dimensional structure and can perform either known or unknown function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, loci (s) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, Ribosome RNA, ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe, and primer. Polynucleotides can include modified nucleotides, such as methylated nucleotides and nucleotide analogs. Modifications to the nucleotide structure, if present, can be made before or after assembly of the polymer. The nucleotide sequence can include non-nucleotide components. The polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.

"Subject" as used herein includes any animal that exhibits or is at risk of exhibiting a disease or symptom that can be treated with the agents of the invention. Suitable subjects include laboratory animals (such as mice, rats, rabbits or guinea pigs), livestock and domestic animals or pets (such as cats or dogs). Includes non-human primates and preferably human patients.

"Substantially" or "essentially" means abundant or significant amounts, quantities, sizes; almost entirely or completely; for example, 95% or more of a given quantity. To do.

A "therapeutic agent" can, when administered in a therapeutically effective amount to a subject (eg, preferably a mammal, more preferably a human), result in the treatment of a disease or condition as defined below. Refers to that compound.

A "therapeutically effective amount" or "therapeutically effective dose", when administered to a subject (eg, preferably a mammal, more preferably a human), is as defined below for the disease or condition in the animal. Refers to the amount of compound of the invention sufficient to provide treatment. The amount of a compound of the invention that constitutes a "therapeutically effective amount" will vary depending on the compound, condition and severity thereof, mode of administration, and age of the animal to be treated, but those skilled in the art will own it. It can be determined by convention by taking into account knowledge and the present disclosure.

"Treatment" or "treatment" herein covers the treatment of a subject having the disease or condition of interest, preferably in humans, (i) in particular the subject. To prevent or prevent the occurrence of a disease or condition in a subject if it has a predisposition to the condition but has not yet been diagnosed with the condition; (ii) Inhibiting the disease or condition, ie, its It includes suppressing the onset; (iii) alleviating the disease or condition, i.e. causing a regression of the disease or condition; or (iv) alleviating the symptoms caused by the disease or condition. As used herein, the terms "disease," "disorder," and "condition" can be used interchangeably, or a particular disease, injury, or condition may not have a known causative agent. It may differ in that it may differ (as the etiology has not yet been elucidated), and therefore it is not yet recognized as an injury or disease, but is simply recognized as an unwanted condition or syndrome, in which case. A set of higher or lower symptomatic specificities has been identified by clinicians.

Abstract The present invention relates, among other things, to autologous and / or allogeneic B cells modified by the introduction of nucleic acids to produce follistatin, and also (eg, diseases, disorders, or conditions, such as muscular dystrophy, etc.) It relates to a method of administering modified B cells (for treating muscular disorders). In some embodiments, the terms "manipulated B cell", "genetically engineered B cell", "modified B cell" and "genetically modified B cell" are one or more nucleic acids for producing follistatin ( Used interchangeably herein to refer to a B cell that has been modified to contain, for example, a transgene) (eg, a transgene that allows expression of a follistatin polypeptide, such as a therapeutic follistatin polypeptide). Has been done. Specifically, the modified B cells can be administered as a single dose or multiple doses.

Therefore, the methods for administering the modified B cell compositions described herein are useful for long-term in vivo delivery and expression of follistatin. The present disclosure relates to methods for achieving sufficient enrichment and number of follistatin-producing cells, as well as sufficient levels of follistatin in vivo, while ensuring product safety in general.

As used herein, the terms "long-term in vivo survival" and "long-term survival" refer to the survival of modified B cells as described herein for 10 days or longer after administration in a subject. Long-term survival can be measured on a daily, weekly or even yearly basis. In one embodiment, the majority of modified B cells are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 after administration. , 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 days or more Also survives in vivo for a long time. In one embodiment, the majority of modified B cells are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 after administration. , 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 , 45, 46, 47, 48, 49, 50, 51, 52 weeks or longer, in vivo. In another embodiment, the modified B cells are 1,1.5,2,2.5,3,3.5,4,4.5,5,6,7,8,9,10,11,12. , 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 years or longer, in vivo. Moreover, the modified B cells described herein can survive in vivo for 10 days or longer, but the majority of modified B cells are 1, 2, 3, 4 after administration. It is understood that they survive in vivo for 5, 6, 7, 8, 9 days or longer. Therefore, it is contemplated that the modified B cells described herein are useful for short-term treatment (eg, 4 days) and long-term treatment (eg, 30 days or longer).

B cells After leaving the bone marrow, B cells act as antigen-presenting cells (APCs) to translocate antigens internally. Antigen is taken up and processed by B cells via receptor-mediated endocytosis. The antigen is processed into an antigen peptide, loaded onto the MHC II molecule, and presented to the extracellular surface of B cells against CD4 + T helper cells. Such T cells bind to MHC ll / antigen molecules and cause B cell activation. After stimulation with T cells, activated B cells begin to differentiate into more specialized cells. Germinal center B cells can differentiate into long-lived memory B cells or plasma cells. In addition, secondary immune stimulation can result in memory B cells that give rise to additional plasma cells. Prior to the formation of plasma cells from either memory or non-memory B cells, the formation of precursor plasma blasts that ultimately differentiate into plasma cells that produce large amounts of antibody occurs (eg, Trends Immunol). June 2009; Volume 30 (No. 6): pp. 277-285; Nature Reviews, 2005, Volume 5: pp. 231-242). Plasma blasts secrete more antibodies than B cells, but less than plasma cells. It divides rapidly, translocates the antigen internally, and continues to present the antigen to T cells. Plasma blasts have the ability to migrate to chemokine-producing sites (eg, in the bone marrow), thereby allowing them to differentiate into long-lived plasma cells. Ultimately, plasma blasts can either die after remaining as plasma blasts for several days or can invariably differentiate into mature, fully differentiated plasma cells. Specifically, plasmablasts capable of homing into tissues containing a plasma cell survival niche (eg, in the bone marrow) are long-lived plasma cells capable of continuing to secrete high levels of protein for several years. Can replace resident plasma cells to become.

B cells used in the methods described herein (eg, for expressing follistatin) include pan-B cells, memory B cells, plasma blasts, and / or plasma cells. In one embodiment, the modified B cell is a memory B cell (eg, modified to express follistatin). In one embodiment, the modified B cells are plasmablasts (eg, modified to express follistatin). In one embodiment, the modified B cells are plasma cells (eg, modified to express follistatin).

Terminally differentiated plasma cells typically do not express common pan-B cell markers such as CD19 and CD20, but express relatively few surface antigens. Plasma cells express CD38, CD78, CD138 and interleukin-6 receptor (IL-6R) and lack expression of CD45, thus identifying plasma cells using these markers, for example by flow cytometry. can do. Since naive B cells are CD27-, memory B cells are CD27 +, and plasma cells are CD27 ++, CD27 is also an excellent marker for plasma cells. Memory B cell subsets can also express surface IgG, IgM and IgD, while plasma cells do not express these markers on the cell surface. CD38 and CD138 are expressed at high levels in plasma cells (Wikipedia, The Free Encyclopedia. "Plasma cell" page version ID: 404969441; Date of latest revision: December 30, 2010, 09:54 UTC, 2011. See January 4, search; Jourdan et al., Blood. December 10, 2009; Volume 114 (No. 25): pp. 5173-81; Trends Encyclopedia. June 2009; Volume 30 (No. 6): 277-285. Pages; Nature Reviews, 2005, Volume 5: pp. 231-242; Nature Med. 2010, Volume 16: pp. 123-129; ). Molecular Wikipedia of B cells. Amsterdam: Elsevier, pp. 189-191; Bertil Glader; Greer, John G .; John Foerster; Rodgers, Georg Volume 2, Set. Hagerston, MD: Lippincott Williams & Wilkins., P. 347; Walport, Mark; Murphy, Kennes; Juneway, Charles; Plasma, December, December, 2008. , Pp. 387-388; Rawtron AC (May 2006) "Immunophenotyping of plasma cells", also see Curr Protocol Cytom).

"Static" as used herein refers to a cellular state in which cells are not actively proliferating.

"Activated" as used herein refers to a cellular state in which cells are actively proliferating and / or producing cytokines in response to stimuli.

The terms "differentiate" and "differentiate" as used herein refer to a change in cell phenotype from one cell type or state to another. For example, memory B cells that migrate to plasma cells are differentiated.

The term "subject" is intended to include living organisms capable of eliciting an adaptive immune response (eg, mammals). Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. In one embodiment, the subject is a human. B cells can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, cord blood, tissue from the site of infection, spleen tissue and tumors. In a preferred embodiment, the source of B cells is PBMC. In certain embodiments of the present disclosure, any number of B cell lines available in the art can be used.

In certain embodiments of the methods described herein, B cells are isolated from Ficoll ™, a copolymer of sucrose and epichlorohydrin that can be used in the preparation of high density solutions, etc. It can be obtained from a unit of blood taken from a subject using any number of techniques known to those of skill in the art. In one preferred embodiment, apheresis or leukocyte apheresis results in cells derived from the individual's circulating blood. Aferesis products typically contain lymphocytes, erythrocytes and platelets, including T cells, monocytes, granulocytes, B cells and other nucleated white blood cells. In one embodiment, the cells collected by apheresis can be washed to remove the plasma fraction and the cells can be placed in a suitable buffer or medium for subsequent processing steps. In one embodiment of the method described herein, cells are washed with phosphate buffered saline (PBS). In an alternative embodiment, the wash solution can lack calcium, magnesium, or many, if not all, divalent cations. As will be readily appreciated by those skilled in the art, the washing steps are known to those of skill in the art, such as by using a semi-automatic "flow-through" centrifuge (eg, Cobe 2991 cell processor) according to the manufacturer's instructions. It can be achieved by the method. After washing, the cells can be resuspended in various biocompatible buffers, such as PBS. Alternatively, the unwanted constituents of the apheresis sample can be removed and the cells can be resuspended directly in the culture medium.

B cells can be isolated from peripheral blood or leukocyte apheresis using techniques known in the art. For example, PBMCs can be isolated using FICOLL ™ (Sigma-Aldrich, St Louis, MO) and CD19 + B cells can be obtained from the Rosette Tetramer Complex System (StemCell Technologies, Vancouver, Canada) or. Purification can be performed by negative or positive selection using any of a variety of antibodies known in the art, such as MACS ™ MicroBed Technology (Miltenyi Biotec, San Diego, CA). In certain embodiments, Memory B cells are isolated as described by Jordan et al. (Blood, December 10, 2009; Vol. 114 (No. 25): pp. 5173-81). For example, after removal of CD2 + cells using anti-CD2 magnetic beads, CD19 + CD27 + Memory B cells can be sorted by FACS. Bone marrow plasma cells (BMPC) can be purified using anti-CD138 magnetic microbead sorting or other similar methods and reagents. Human B cells can be isolated using, for example, CD19 MicroBead, human (Miltenyi Biotec, San Diego, CA). Human Memory B cells can be isolated using, for example, Memory B cell isolation kits, humans (Miltenyi Biotec, San Diego, CA).

Other isolation kits are commercially available, such as the R & D Systems MagCellect human B cell isolation kit (Minneapolis, MN). In certain embodiments, resting B cells can be prepared by sedimentation in a discontinuous Percoll gradient, as described in (Defranco et al. (1982) J. Exp. Med. 155: 1523). ..

In one embodiment, PBMCs are obtained from blood samples using gradient-based purification (eg, FICOLL ™). In another embodiment, PBMCs are obtained from apheresis-based harvesting. In one embodiment, B cells are isolated from PBMCs by isolating pan-B cells. Isolation steps can utilize positive and / or negative selection. In one embodiment, the negative selection comprises T cell depletion using anti-CD3 conjugated microbeads, thereby providing a T cell depletion fraction. In a further embodiment, Memory B cells are isolated from pan-B cell or T cell depleted fractions by positive selection for CD27.

In one particular embodiment, Memory B cells are isolated by unwanted cell depletion and subsequent positive selection by CD27 MicroBead. Unwanted cells such as T cells, NK cells, monocytes, dendritic cells, granulocytes, platelets and erythrocyte lineage cells are biotinylated antibodies against CD2, CD14, CD16, CD36, CD43 and CD235a (glycophorin A). It can be depleted using cocktails as well as anti-biotin MicroBed.

In one embodiment, switched memory B cells are obtained. "Switched memory B cells" or "switched B cells" as used herein refer to B cells that have undergone isotype class switching. In one embodiment, the switched Memory B cells are positively selected for IgG. In another embodiment, switched memory B cells are obtained by depleting IgD and IgM expressing cells. Switched Memory B cells can be isolated using, for example, switched Memory B cell kits, humans (Miltenyi Biotec, San Diego, CA).

For example, in one particular embodiment, non-target cells can be labeled with a cocktail of biotinylated CD2, CD14, CD16, CD36, CD43, CD235a (glycophorin A), anti-IgM and anti-IgD antibodies. Such cells can then be magnetically labeled with anti-biotin MicroBead. Highly pure switched memory B cells can be obtained by depletion of magnetically labeled cells.

In a further embodiment, a promoter sequence derived from a gene specific to Memory B cells, such as the CD27 gene (or another gene that is specific to Memory B cells and is not expressed in naive B cells), is the presence of, for example, methotrexate. It is used to drive the expression of selectable promoters, such as mutated dihydrofolate reductase, which allows for positive selection of Memory B cells below. In another embodiment, a promoter sequence derived from a pan-B cell gene, such as the CD19 gene, is a selectable marker, such as a mutated dihydrofolate reductase that allows positive selection of memory B cells in the presence of methotrexate. Used to drive the expression of. In another embodiment, T cells are depleted using CD3 or by the addition of cyclosporine. In another embodiment, CD138 + cells are isolated from pan-B cells by positive selection. In yet another embodiment, CD138 + cells are isolated from PBMCs by positive selection. In another embodiment, CD38 + cells are isolated from pan-B cells by positive selection. In yet another embodiment, CD38 + cells are isolated from PBMCs by positive selection. In one embodiment, CD27 + cells are isolated from PBMCs by positive selection. In another embodiment, Memory B cells and / or plasma cells are selectively expanded from PBMCs using in vitro culture methods available in the art.

Culturing B cells in vitro B cells, such as memory B cells, can be cultured using in vitro methods for activating B cells and differentiating them into plasma cells and / or plasma cells. As will be recognized by those of skill in the art, plasma cells can be identified by cell surface protein expression patterns using standard flow cytometry methods. For example, terminally differentiated plasma cells express relatively few surface antigens and do not express common pan-B cell markers such as CD19 and CD20. Instead, plasma cells can be identified by the expression of CD38, CD78, CD138 and IL-6R, as well as the lack of expression of CD45. Since naive B cells are CD27-, memory B cells are CD27 +, and plasma cells are CD27 ++, CD27 can also be used to identify plasma cells. Plasma cells express high levels of CD38 and CD138.

In one embodiment, the B cells are CD138-memory B cells. In one embodiment, the B cells are CD138 + plasma cells. In one embodiment, B cells are activated and have a cell surface phenotype of CD138-, CD27 +.

In one embodiment, the B cells are CD20-, CD138-memory B cells. In one embodiment, the B cells are CD20-, CD138 + plasma cells. In one embodiment, B cells are activated and have a cell surface phenotype of CD20-, CD138-, CD27 +.

In one embodiment, the B cells are CD20-, CD38-, CD138-memory B cells. In one embodiment, the B cells are CD20-, CD38 +, CD138 + plasma cells. In one embodiment, B cells are activated and have a cell surface phenotype of CD20-, CD38-, CD138-, CD27 +.

In one embodiment, B cells are in contact with one or more B cell activating factors, such as any of a variety of cytokines, growth factors or cell lines known to activate and / or differentiate B cells. (See, eg, Fluckiger et al., Blood 1998, Vol. 92: pp. 4509-4520; Luo et al., Blood 2009, Vol. 113: pp. 1422-1431). Such factors are IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL. -12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24 , IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34 and IL-35, IFN-γ, IFN -Α, IFN-β, IFN-δ, C-type chemokines XCL1 and XCL2, CC-type chemokines (to date, including CCL1 to CCL28) and CXC-type chemokines (to date, including CXCL1 to CXCL17), and TNF. Members of the superfamily (eg, TNF-α, 4-1 BB ligand, B cell activator (BLyS), FAS ligand, sCD40L (including multimeric versions of sCD40L; eg, multiple sCD40L molecules grouped together) From the group consisting of histidine-tagged soluble recombinant CD40L), phosphotoxins, OX40L, RANKL, TRAIL), CpG, and other trol-like receptor agonists (eg, CpG) in combination with anti-poly-histidine mAbs. You can choose, but you are not limited to these.

B cell activating factor can be added to in vitro cell cultures at various concentrations to achieve the desired outcome (eg, expansion or differentiation). In one embodiment, the B cell activating factor is utilized in the expansion and proliferation of B cells in culture. In one embodiment, the B cell activating factor is utilized in the differentiation of B cells in culture. In another embodiment, the B cell activating factor is utilized in both the expansion and differentiation of B cells in culture. In one embodiment, the B cell activating factor is provided at the same concentration for expansion and differentiation. In another embodiment, the B cell activating factor is provided at a first concentration for expansion and a second concentration for differentiation. B cell activating factor can be used in 1) B cell expansion and proliferation and cannot be used in B cell differentiation. 2) Use in B cell differentiation and not in B cell expansion and proliferation. It is contemplated that it can be used, or 3) it can be utilized in the expansion and differentiation of B cells.

For example, in some embodiments, B cells are one or more B cell activating factors selected from CD40L, IL-2, IL-4 and IL-10 for B cell expansion. Is cultured with a B cell culture medium containing. In one embodiment, B cells are cultured with 0.25-5.0 μg / ml CD40L. In one embodiment, the concentration of CD40L is 0.5 μg / ml. In one embodiment, a cross-linking agent (such as an anti-HIS antibody combined with HIS-tagged CD40L) is used to make a multimer of CD40L. In one embodiment, the molecules of CD40L are covalently linked or held together using a protein multimerization domain (eg, the Fc region of IgG or the leucine zipper domain). In one embodiment, the CD40L is conjugated to beads. In one embodiment, CD40L is expressed from feeder cells. In one embodiment, B cells are cultured with 1-10 ng / ml IL-2. In one embodiment, the concentration of IL-2 is 5 ng / ml. In one embodiment, B cells are cultured with 1-10 ng / ml IL-4. In one embodiment, the concentration of IL-4 is 2 ng / ml. In one embodiment, B cells are cultured with 10-100 ng / ml IL-10. In one embodiment, the concentration of IL-10 is 40 ng / ml.

In one embodiment, the B cell is one or more B cells selected from CD40L, IL-2, IL-4, IL-10, IL-15 and IL-21 for the expansion and proliferation of B cells. It is cultured with a B cell culture medium containing a cell activating factor. In one embodiment, B cells are cultured with 0.25-5.0 μg / ml CD40L. In one embodiment, the concentration of CD40L is 0.5 μg / ml. In one embodiment, a cross-linking agent (such as an anti-HIS antibody combined with HIS-tagged CD40L) is used to make a multimer of CD40L. In one embodiment, the molecules of CD40L are covalently linked or held together using a protein multimerization domain (eg, the Fc region of IgG or the leucine zipper domain). In one embodiment, the CD40L is conjugated to beads. In one embodiment, CD40L is expressed from feeder cells. In one embodiment, B cells are cultured with 1-10 ng / ml IL-2. In one embodiment, the concentration of IL-2 is 5 ng / ml. In one embodiment, B cells are cultured with 1-10 ng / ml IL-4. In one embodiment, the concentration of IL-4 is 2 ng / ml. In one embodiment, B cells are cultured with 10-100 ng / ml IL-10. In one embodiment, the concentration of IL-10 is 40 ng / ml. In one embodiment, B cells are cultured with 50-150 ng / ml IL-15. In one embodiment, the concentration of IL-15 is 100 ng / ml. In one embodiment, B cells are cultured with 50-150 ng / ml IL-21. In one embodiment, the concentration of IL-21 is 100 ng / ml. In certain embodiments, B cells are cultured with a B cell culture medium containing CD40L, IL-2, IL-4, IL-10, IL-15 and IL-21 for B cell expansion. ..

For example, in one embodiment, the B cells are B cells that contain the B cell activating factors CD40L, IL-2, IL-4, IL-10, IL-15 and IL-21 for the expansion and proliferation of B cells. Cultured with culture medium, CD40L is crosslinked with a cross-linking agent to produce multimers of CD40L. Such a culture system can be maintained for the entire culture period (eg, a 7-day culture period) so that B cells express the transgene of interest (eg, an exogenous polypeptide such as FST). It has been transfected or otherwise manipulated. The transgene can be integrated into B cells (eg, by viral or non-viral vector). The transgene can be expressed in B cells by using a transposon. The transgene can be expressed in B cells for targeted integration of the transgene into the B cell genome. Targeted integration can be by homologous recombination. Homologous recombination can occur during nuclease-induced double-strand breaks. The nuclease can be, for example, a zinc finger nuclease, a TALE-nuclease (TALEN), a meganuclease (eg, a homing endonuclease), or can be from the CRISPR / CAS9-nuclease system.

In another example, in some embodiments, B cells are CD40L, IFN-α, IL-2, IL-6, IL-10, IL-15, IL-21 and for B cell differentiation. It is cultured with a B cell culture medium containing one or more B cell activating factors selected from P-class CpG oligodeoxynucleotides (p-ODN). In one embodiment, B cells are cultured with 25-75 ng / ml CD40L. In one embodiment, the concentration of CD40L is 50 ng / ml. In one embodiment, B cells are cultured with 250-750 U / ml IFN-α. In one embodiment, the concentration of IFN-α is 500 U / ml. In one embodiment, B cells are cultured with 5-50 U / ml IL-2. In one embodiment, the concentration of IL-2 is 20 U / ml. In one embodiment, B cells are cultured with 25-75 ng / ml IL-6. In one embodiment, the concentration of IL-6 is 50 ng / ml. In one embodiment, B cells are cultured with 10-100 ng / ml IL-10. In one embodiment, the concentration of IL-10 is 50 ng / ml. In one embodiment, B cells are cultured with 1-20 ng / ml IL-15. In one embodiment, the concentration of IL-15 is 10 ng / ml. In one embodiment, B cells are cultured with 10-100 ng / ml IL-21. In one embodiment, the concentration of IL-21 is 50 ng / ml. In one embodiment, B cells are cultured with 1-50 μg / ml p-ODN. In one embodiment, the concentration of p-ODN is 10 μg / ml.

In one embodiment, B cells are contacted or cultured on feeder cells. In one embodiment, the feeder cell is a stroma cell line, eg, a mouse stroma cell line S17 or MS5. In another embodiment, the isolated CD19 + cells are one or more B cells, such as IL-10 and IL-4, in the presence of fibroblasts expressing the CD40-ligand (CD40L, CD154). Cultured with activator cytokines. In one embodiment, the CD40L is provided bound to a surface such as a tissue culture plate or beads. In another embodiment, purified B cells are CD40L in the presence or absence of feeder cells, as well as IL-10, IL-4, IL-7, p-ODN, CpG DNA, IL-2, IL. It is cultured with one or more cytokines or factors selected from -15, IL6 and IFN-α.

In another embodiment, the B cell activating factor is provided by transfection into B cells or other feeder cells. In this context, one or more factors that promote the differentiation of B cells into antibody-secreting cells and / or one or more factors that promote longevity of antibody-producing cells can be used. Such factors include, for example, anti-apoptotic factors such as Blimp-1, TRF4, Bcl-xl or Bcl5, or constitutively active variants of the CD40 receptor. In addition, factors that promote the expression of downstream signaling molecules, such as TNF receptor-related factors (TRAFs), can also be used in B cell activation / differentiation. In this regard, the cell activation, cell survival and anti-apoptotic functions of the TNF receptor superfamily are largely mediated by TRAF1-6 (eg, RH Arch et al., Genes Dev. 12 (1998). Year) (see pages 2821 to 2830). Downstream effectors of TRAF signaling include transcription factors in the NF-κB and AP-1 families that can turn on genes involved in various aspects of cellular and immune function. Furthermore, activation of NF-κB and AP-1 has been shown to result in cell protection from apoptosis by transcription of anti-apoptotic genes.

In another embodiment, a protein from Epstein-Barr virus (EBV) is used for B cell activation and / or differentiation, or to promote longevity of antibody-producing cells. Examples of EBV-derived proteins include EBNA-1, EBNA-2, EBNA-3, LMP-1, LMP-2, EBER, miRNA, EBV-EA, EBV-MA, EBV-VCA and EBV-AN. Not limited to.

In certain embodiments, contact of a B cell activator with a B cell using the methods provided herein is, among other things, cell proliferation (ie, proliferation), activated mature B. It results in modulation of the lgM + cell surface phenotype to a cell-consistent phenotype, Ig secretion, and isotype switching. CD19 + B cells can be isolated using known and commercially available cell separation kits such as Miltenyi Biotec, Bergisch Gladbach, Germany). In certain embodiments, CD40L fibroblasts are irradiated prior to use in the methods described herein. In one embodiment, B cells are cultured in the presence of one or more of IL-3, IL-7, Flt3 ligand, thrombopoietin, SCF, IL-2, IL-10, G-CSF and CpG. To. In certain embodiments, the method, in combination with transformed stroma cells (eg, MS5) that provide low levels of tethered CD40L and / or CD40L bound to a plate or bead, among the factors described above. Includes the step of culturing B cells in the presence of one or more species.

As described above, B cell activating factor induces B cell expansion, proliferation or differentiation. Thus, B cells are contacted with one or more of the B cell activating factors listed above to obtain an expanded cell population. The cell population can proliferate prior to transfection. Alternatively or further, the cell population can proliferate after transfection. In one embodiment, expansion of the B cell population comprises culturing cells with IL-2, IL-4, IL-10 and CD40L (eg, Nero et al., PLos ONE, 2012, Volume 7 (12). No.): See page 51946). In one embodiment, the expansion of the B cell population comprises culturing cells with IL-2, IL-10, CpG and CD40L. In one embodiment, the expansion of the B cell population comprises culturing cells with IL-2, IL-4, IL-10, IL-15, IL-21 and CD40L. In one embodiment, expansion of the B cell population comprises culturing cells with IL-2, IL-4, IL-10, IL-15, IL-21 and multimerized CD40L.

In another embodiment, the expansion of the B cell population is induced and / or enhanced by a transgene introduced into B cells. For example, it contains a recombinant or engineered receptor that induces a cell signaling pathway (eg, signaling downstream of CD40) when bound to that ligand (eg, soluble or surface-expressing ligand). B cells. In one embodiment, B cells overexpress CD40 due to the expression of the CD40 transgene. In another embodiment, B cells express engineered receptors, including, for example, recombinantly engineered antibodies. In one embodiment, the engineered receptor is similar to a chimeric antigen receptor (CAR) and comprises a fusion protein of scFv and the intracellular signaling portion of the B cell receptor (eg, CD40).

In one embodiment, the expansion of the B cell population is induced and / or enhanced by small molecule compounds added to the cell culture. For example, compounds that bind to and dimerize CD40 can be used to induce the CD40 signaling pathway.

As is known to those of skill in the art, any of a variety of culture media can be used in this method (see, eg, Currant Protocols in Cell Culture, 2000-2009, John Wiley & Sons, Inc.). In one embodiment, media for use in the methods described herein includes, but is not limited to, Iskov modified Dulbecco medium (containing or not containing fetal bovine serum or other suitable serum). .. Exemplary media include, but are not limited to, IMDM, RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15 and X-Vivo 20. In a further embodiment, the medium is a surfactant, antibody, plasmanate or reducing agent (eg, N-acetyl-cysteine, 2-mercaptoethanol), one or more antibiotics, and / or Additives such as insulin, transferrin, sodium selenite and cyclosporine can be included. In some embodiments, IL-6, soluble CD40L and crosslink enhancer can also be used.

B cells are cultured under conditions and for a sufficient period of time to achieve the desired differentiation and / or activation. In certain embodiments, B cells are 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% of B cells. 70%, 75%, 80%, 85%, 90%, 95% or even 100% are cultured under conditions and for a sufficient period of time such that they are differentiated and / or activated as desired. In one embodiment, B cells are activated and differentiated into a mixed population of plasmablasts and plasma cells. As will be recognized by those skilled in the art, plasmablasts and plasma cells are ^{expressed in one or more of CD38, CD78, IL-6R, CD27 high} and CD138, and / or of CD19, CD20 and CD45. The lack or reduction of expression of one or more can be identified by cell surface protein expression patterns using standard flow cytometry methods as described elsewhere herein. As will be appreciated by those skilled in the art, memory B cells are generally CD20 +, CD19 +, CD27 +, CD38-, while early plasma blasts are CD20-, CD19 +, CD27 ++, CD38 ++. In one embodiment, the cells cultured using the methods described herein are CD20-, CD38 +, CD138-. In another embodiment, the cells have the CD20-, CD38 +, CD138 + phenotypes. In certain embodiments, cells are cultured for 1-7 days. In a further embodiment, the cells are cultured for 7, 14, 21 days or longer. Therefore, the cells are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 under appropriate conditions. , 21, 22, 23, 24, 25, 26, 27, 28, 29 days or longer. The cells are reseeded and media and supplements can be added or replaced as needed using techniques known in the art.

In certain embodiments, B cells are at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% of cells. 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% are differentiated and activated to produce Ig, and / Or culture under conditions and for a sufficient period of time to express the transgene.

B cell activation is induced ^{by techniques such as 3} H-uridine uptake into RNA (RNA synthesis increases as B cells differentiate), or by measuring DNA synthesis associated with cell proliferation ³ H-thymidine uptake. Can be measured by. In one embodiment, interleukin-4 (IL-4) can be added to the culture medium at a concentration suitable for enhancing B cell proliferation (eg, about 10 ng / ml).

Alternatively, B cell activation is measured as a function of immunoglobulin secretion. For example, CD40L is added to resting B cells along with IL-4 (eg, 10 ng / ml) and IL-5 (eg, 5 ng / ml), or other cytokines that activate B cells. Flow cytometry can also be used to measure cell surface markers typical of activated B cells. For example, Civin CI, Looken MR, Int'l J. et al. Cell Cloning 987; Volume 5: pp. 1-16; Loken, MR et al., Flow Cytometry characterization of Erythroid, Lymphoid and Monomyeroid Lineages in Normal Human Body Eds., Academic Press, New York 1992; pp. 31-42; and LeBein TW et al., Leukemia 1990; Volume 4: pp. 354-358.

Additional volumes of culture medium may be added after culturing for an appropriate period, eg, 2, 3, 4, 5, 6, 7, 8, 9 days or longer, generally around 3 days, etc. it can. Supernatants from individual cultures were collected at various time points in the culture, Noelle et al. (1991) J. Mol. Immunol. It can be quantified for IgM and IgG1 as described in Volumes 146: 1118 to 1124. In one embodiment, cultures are collected and measured for expression of the introduced gene of interest using flow cytometry, enzyme-linked immunosorbent assay (ELISA), ELISA or other assays known in the art. To.

In another embodiment, ELISA is used to measure antibody isotype production, such as IgM, or the product of the transgene of interest. In certain embodiments, IgG determination is made using commercially available antibodies such as goat anti-human IgG as capture antibody, followed by a variety of biotinylated goat anti-human Ig, streptavidin alkaline phosphatase and substrates. Detection is made using one of the appropriate detection reagents.

In certain embodiments, the number of B cells is 1, 10, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, which is the number of B cells at the start of culture. Incubate under conditions and for a sufficient period of time to be 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 times or more. In one embodiment, the number of cells is 10 to 1000 times the number of B cells at the start of culture (including consecutive integers therein). For example, the expanded B cell population is at least 10 times larger than the initially isolated B cell population. In another embodiment, the expanded B cell population is at least 100 times larger than the initially isolated B cell population. In one embodiment, the expanded B cell population is at least 500 times larger than the initially isolated B cell population.

Manipulating B Cells In various embodiments, the present disclosure transfects B cells, infects B cells, or otherwise transgenes (eg, transgenes) such that the transgene is expressed in B cells. , A method for incorporating a follistatin transgene) into B cells. Any of the integration methods described herein or known in the art can be utilized to express follistatin in B cells in some embodiments.

In one embodiment, the genetically modified B cells are transfected with the transgene. In certain embodiments, the genetically modified B cells are associated with a follistatin transgene.

Exemplary methods for transfecting cells are provided in WO2014 / 152832 and WO2016 / 100932, both of which are incorporated herein by reference in their entirety. Transfection of B cells can be accomplished using any of the various methods available in the art for introducing DNA or RNA into B cells. Suitable techniques include calcium phosphate transfection, DEAE-dextran, electroporation, pressure-mediated transfection or "cell squeezing" (eg, CellSqueeze microfluidic system, SQZ Biotechnology), nanoparticle-mediated or liposome-mediated. Sex transfection and transduction using retroviruses or other viruses, such as vaccinia, can be included. For example, Graham et al., 1973, Virology 52: 456; Sambrook et al., 2001, Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Biology, Elsevier, 1986. 1981, Gene Vol. 13, p. 197; US5,124,259; US5,297,983; US5,283,185; US5,661,018; US6,878,548; US7,799,555; US8,551 780; and US8,633,029. An example of a commercially available electroporation technique suitable for B cells is the Nucleofector ™ transfection technique.

Transfection can be performed before or during in vitro culture of isolated B cells in the presence of one or more of the activations and / or differentiation factors described above. For example, cells are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 in vitro cultures. , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39 days. In one embodiment, cells are transfected on days 1, 2 or 3 of in vitro culture. In certain embodiments, cells are transfected on day 2. For example, cells are electroporated on day 2 of in vitro culture, for example for delivery of plasmids, transposons, minicircles or self-replicating RNA. In another embodiment, cells are transfected on days 4, 5, 6 or 7 of in vitro culture. In certain embodiments, cells are transfected on day 6 of in vitro culture. In another embodiment, cells are transfected on day 5 of in vitro culture.

In one embodiment, cells are transfected or otherwise engineered prior to activation (eg, by targeted integration of a follistatin transgene). In another embodiment, cells are transfected or otherwise engineered during activation (eg, by targeted integration of a follistatin transgene). In one embodiment, cells are transfected or otherwise engineered after activation (eg, by targeted integration of a follistatin transgene). In one embodiment, cells are transfected or otherwise engineered prior to differentiation (eg, by targeted integration of a follistatin transgene). In another embodiment, cells are transfected or otherwise manipulated during differentiation (eg, by targeted integration of a follistatin transgene). In one embodiment, cells are transfected or otherwise manipulated after differentiation (eg, by targeted integration of a follistatin transgene).

In one embodiment, a non-viral vector is used to deliver DNA or RNA (eg, DNA or RNA containing a sequence encoding a follistatin polypeptide) to Memory B cells and / or plasma cells. For example, systems that can facilitate transfection of memory B cells and / or plasmid cells without the need for a viral integration system are transposons (eg, sleeping beauty or other transposon systems such as Piggybac), zinc finger nucleases. (ZFN), transcriptional activator-like effector nucleases (TALENs), clustered and regularly arranged short circular sequence repeats (CRISPR), meganucleases, minicircles, replicons, artificial chromosomes (eg, bacterial artificial chromosomes, mammals) Animal artificial chromosomes and yeast artificial chromosomes), plasmids, cosmids, and bacteriophages are included without limitation.

In some embodiments, such non-virus dependent vector systems can also be delivered by viral vectors known in the art or described below. For example, in some embodiments, the viral vector (eg, retrovirus, lentivirus, adenovirus, adeno-associated virus) is one or more non-viral vectors (eg, the zinc finger nuclease (ZFN) described above). Transcriptional activator-like effector nucleases (TALENs), one or more clustered, well-arranged, short circular sequence repeat (CRISPR) meganucleases, etc.), or targeted integration can be facilitated. Utilized for delivery of any other enzyme / complementary vector, polynucleotide and / or polypeptide. Thus, in some embodiments, cells (eg, memory B cells and / or plasma cells, etc., B cells) have an exogenous sequence (eg, a sequence encoding a follistatin polypeptide) by a targeted integration method. It can be manipulated to manifest. Such methods are known in the art and include the step of cleaving an endogenous locus in a cell using one or more nucleases (eg, ZFN, TALEN, CRISPR / Cas, meganuclease). Can include the step of administering the follistatin transgene into the cell so that it is integrated into the endogenous locus and expressed intracellularly. The follistatin transgene may be included in the donor sequence that integrates into the DNA of the host cell at or near the cleavage point by the nuclease.

Integration of exogenous sequences (eg, sequences encoding follistatin polypeptides) can occur by recombination. As will be apparent to those of skill in the art, "recombination" includes, but is not limited to, donor capture and homologous recombination by non-homologous end binding (NHEJ), genetic information between two polynucleotides. Refers to the process of exchange. The recombination can be a homologous recombination. For the purposes of the present disclosure, "homologous recombination (HR)" refers to, for example, a specialized form of such exchange performed during repair of double-strand breaks in cells by a homologous directional repair mechanism. Point to. This process utilizes nucleotide sequence homology, whereby a "donor" molecule (eg, a donor polynucleotide sequence or a donor vector containing such a sequence) experiences a "target" molecule (ie, double-strand breaks). It is used by the DNA repair mechanism of cells as a template for repairing (molecules), and these means cause the transfer of genetic information from the donor to the target. In some embodiments of HR-directed integration, the donor molecule can contain at least two regions of homology to the genome (“homology arm”). In some embodiments, the homology arm can be, for example, at least 50-100 base pairs in length. The homology arm can have substantial DNA homology to the region of genomic DNA adjacent to the cleavage site where targeted integration should occur. The homology arm of the donor molecule can be flanked by DNA that should be integrated into the target genome or target DNA locus (eg, including DNA encoding follistatin). Chromosome cleavage followed by repair using the homologous region of plasmid DNA as a template can result in the transfer of intervening transgenes flanked by homology arms into the genome. For example, Koller et al. (1989) Proc. Nat'l. Acad. Sci. USA Vol. 86 (No. 22): 8927-893; See Thomas et al. (1986) Cell Vol. 44 (3): 419-428. The frequency of this type of homology directed targeted integration can be increased up to 10 ⁵ times the planned production of double-strand breaks in the vicinity of the target area (Hockemeyer et al (2009) Nature Biotech.27 Volume (9 (No.): pp. 851-857; Lombardo et al. (2007) Nature Biotech. Vol. 25 (No. 11): pp. 1298-1306; Moehle et al. (2007) Proc. Nat'l Acad. Sci. USA Vol. 104 (No. 9). ): pp. 3055-3060; Rouet et al. (1994) Proc. Nat'l Acad. Sci. USA Vol. 91 (No. 13): pp. 6064-6068.

Any nuclease that can mediate targeted cleavage of a genomic locus such that the transgene (eg, foristatin transgene) can be integrated into the genome of the target cell (eg, by recombination, such as HR) , Can be utilized in the manipulation of cells according to the present disclosure (eg, memory B cells or plasmablasts).

Double-strand breaks (DSBs) or nicks can be made by site-specific nucleases such as zinc finger nucleases (ZFNs), TAL effector domain nucleases (TALENs), meganucleases, or engineered crRNA / to guide specific breaks. It can be made using a CRISPR / Cas9 system with a tract RNA (single guide RNA). For example, Burgess (2013) Nature Reviews Genetics, Vol. 14, pp. 80-81, Urnov et al. (2010), Nature 435 (7042): 646, which incorporates their entire disclosures herein by reference for all purposes. Pp. 51; U.S. Patent Application Publication No. 20030232410; No. 20050208489; No. 20050026157; No. 20050064474; No. 20060188897; No. 200909263900; No. 200901017617; No .; see 20110101073 and WO 2007/014275.

In some embodiments, cells (eg, Memory B cells or plasmablasts) are engineered by zinc finger nuclease-mediated targeting integration of donor constructs (eg, follistatin donor constructs). Zinc finger nucleases (ZFNs) are unique due to the coupling of one or more zinc fingers and a "zinc finger DNA binding protein" (ZFP) (or binding domain) that binds DNA in a sequence-specific manner via a nuclease enzyme. It is an enzyme capable of recognizing and cleaving a target nucleotide sequence by sex. ZFNs contain any suitable cleavage domain (eg, a nuclease enzyme) operably linked to a ZFP DNA binding domain and can facilitate site-specific cleavage of the target DNA sequence. It can be formed (see, eg, Kim et al. (1996) Proc Natl Acad Sci USA Vol. 93 (3): pp. 1156-1160). For example, ZFNs can include a FOK1 enzyme or a target-specific ZFP linked to a portion of the FOK1 enzyme. In some embodiments, the ZFNs used in the ZFN-mediated targeting integration approach utilize two separate molecules, each containing a subunit of the FOK1 enzyme bound to each ZFP, where each ZFP is targeted. Having specificity for the DNA sequence adjacent to the cleavage site, when the two ZFPs bind to their respective target DNA sites, the FOK1 enzyme subunits are brought close to each other and bind together to the target cleavage site. Activates the nuclease activity that cleaves. ZFNs have been used for genomic modification in a variety of organisms (eg, U.S. Patent Application Publication Nos. 20030232410; 20050208489; 20050026157; 20050064474, all of which are incorporated herein by reference. No. 20060188987; No. 20060063231; and International Publication WO07 / 014,275). Custom ZFPs and ZFNs are commercially available, for example, from Sigma Aldrich (St. Louis, MO), and such custom ZFNs can also be used to routinely target and cleave any position in the DNA.

In some embodiments, cells (eg, Memory B cells or plasmablasts) are engineered by CRISPR / Cas (eg, CRISPR Cas9) nuclease-mediated integration of donor constructs (eg, follistatin donor constructs). The CRISPR (clustered and regularly arranged short palindromic sequence repeat) / Cas (CRISPR-related) nuclease system is an engineered nuclease system based on a bacterial system that can be used for genomic manipulation. It is based on some of the adaptive immune responses of many and archaea. When a virus or plasmid invades a bacterium, a segment of the invader's DNA is converted to CRISPR RNA (crRNA) by an "immune" response. This crRNA then associates with another type of RNA called tracrRNA via a region of partial complementarity to guide Cas9 nuclease to a region homologous to crRNA in the target DNA called a "protospacer". Cas9 cleaves the DNA to produce a blunt stump in the DSB at the site specified by the 20 nucleotide guide sequence contained within the crRNA transcript. Cas9 requires both crRNA and tracrRNA for site-specific DNA recognition and cleavage. This system is currently being engineered to allow the combination of crRNA and tracrRNA into a single molecule (“single guide RNA”), where the crRNA-corresponding portion of the single guide RNA targets any desired sequence. It can be manipulated to guide Cas9 nucleases (Jinek et al. (2012) Science 337, pp. 816-821, Jinek et al. (2013) eLife Vol. 2: e00471 and David Segal (2013) eLife. Volume 2: see page 3 of e0056). Thus, the CRISPR / Cas system can be engineered to make DSBs at the desired target in the genome, and repair of DSBs can be affected by the use of repair inhibitors that cause an increase in error-prone repairs. .. As will be apparent to those skilled in the art, in addition to Cas9, other CRISPR nucleases are known and are suitable for use in the present invention.

In some embodiments, CRISPR / Cas nuclease-mediated integration utilizes type II CRISPR. Type II CRISPR is one of the most well-characterized systems, performing targeted DNA double-strand breaks in four sequential steps. First, two non-coding RNAs, the pre-crRNA array and the tracrRNA, are transcribed from the CRISPR locus. Second, the tracrRNA hybridizes to the repeating region of the pre-crRNA and mediates the processing of the pre-crRNA into a mature crRNA containing individual spacer sequences. Third, the mature crRNA: tracrRNA complex is an additional requirement for target recognition, the Watson-Crick base between the spacer in crRNA and the protospacer in the target DNA next to the protospacer flanking motif (PAM). Pairing directs Cas9 towards the target DNA. Fourth, Cas9 mediates the cleavage of the target DNA to create double-strand breaks within the protospacer.

Cas9-related CRISPR / Cas systems include a pre-crRNA array containing two RNA non-coding components: tracrRNA and a nuclease guide sequence (spacer) interspersed with the same serial repeats (DRs). In order to achieve genomic manipulation using the CRISPR / Cas system, the function of both of these RNAs must be present (see Kong et al. (2013) Science Express 1 / 10.1126 / science 1231143). .. In some embodiments, tracrRNA and precrRNA are supplied by separate expression constructs or as separate RNAs. In another embodiment, a chimeric RNA is constructed, wherein the engineered mature crRNA (which imparts target specificity) is fused to a tracrRNA (which supplies the interaction with Cas9) and the chimeric cr-RNA- Create a tracrRNA hybrid (also named a single guide RNA). (See Jinek, said section and Kong, said section).

In some embodiments, a single guide RNA containing both crRNA and tracrRNA can be engineered to guide Cas9 nuclease to target any desired sequence (eg, Jinek et al. (2012)). Year) Science Vol. 337, pp. 816-821, Jinek et al. (2013) eLife Vol. 2: e00471, David Segal (2013) eLife Vol. 2: e00563). Thus, the CRISPR / Cas system can be engineered to create a DSB at a desired target in the genome.

Custom CRISPR / Cas systems are commercially available, for example, from Dharmacon (Lafayette, CO), and such custom single-guide RNA sequences can also be used to routinely target and cleave any position in the DNA. .. Single-stranded DNA templates for recombination can be synthesized (eg, by methods known and commercially available in the art for oligonucleotide synthesis) or provided in a vector, eg, a viral vector such as AAV. be able to.

In some embodiments, cells (eg, Memory B cells or plasmablasts) are engineered by TALE-nuclease (TALEN) -mediated targeting integration of donor constructs (eg, follistatin donor constructs). A "TALE DNA binding domain" or "TALE" is a polypeptide containing one or more TALE repeat domains / units. The repetitive domain is involved in the binding of TALE to its cognate target DNA sequence. A single "repeated unit" (also referred to as "repeated") is typically 33-35 amino acids in length and has at least some degree of sequence homology with other TALE repeats within the naturally occurring TALE protein. Is shown. TAL-effectors can contain tandem repeat nuclear localization sequences, acidic transcription activation domains and centrally concentrated domains, with each repeat being approximately 34 amino acids key to the DNA binding specificity of such proteins. Contains. (For example, Schornack S, et al. (2006) J Plant Physiol, Vol. 163 (No. 3): pp. 256-272). TAL effectors depend on the sequences found in tandem repeats containing approximately 102 bp, and the repeats are typically 91-100% homologous to each other (eg, Bonas et al. (1989) MoI Gen Genet Vol. 218: 127-136). page). Artificial transcription factors capable of manipulating such DNA binding iterations to put new combinations and numbers of iterations into proteins to interact with new sequences and activate expression of non-endogenous reporter genes It can be made (eg, Bonas et al. (1989) MoI Gen Genet Vol. 218, pp. 127-136). The engineered TAL protein can be ligated to the FokI cleavage half domain to obtain a TAL effector domain nuclease fusion (TALEN) for cleavage of the target-specific DNA sequence (eg, Christian et al. (2010) Genetics). , EPUB 10.1534 / genetics. 110.120717).

Custom TALENs are commercially available, for example, from Thermo Fisher Scientific (Waltham, MA) and can also customarily target and cleave any position in DNA.

In some embodiments, cells (eg, Memory B cells or plasmablasts) are engineered by meganuclease-mediated targeting integration of donor constructs (eg, follistatin donor constructs). Meganucleases (or "homing endonucleases") are endonucleases that bind to and cleave double-stranded DNA in recognition sequences greater than 12 base pairs. The naturally occurring meganuclease can be a monomer (eg, I-SceI) or a dimer (eg, I-CreI). Naturally occurring meganucleases recognize 15-40 base pair cleavage sites and are generally grouped into four families: the LAGLIDADG family, the GIY-YIG family, the His-Cyst box family and the HNH family. Exemplary homing endonucleases are I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I- Includes CreI, I-TevI, I-TevII and I-TevIII. These recognition sequences are known. US Pat. No. 5,420,032; US Pat. No. 6,833,252; Belfort et al. (1997) Nucleic Acids Res. Volume 25: 3379-3388; Dujon et al. (1989) Gene 82: 115-118; Perler et al. (1994) Nucleic Acids Res. Vol. 22, pp. 1125-1127; Jasin (1996) Trends Genet. Volume 12: pp. 224-228; Gimble et al. (1996) J. Mol. Mol. Biol. Volume 263: pp. 163-180; Argast et al. (1998) J. Mol. Mol. Biol. See also Volume 280: pp. 345-353 and the New England Biolabs Catalog. The term "meganuclease" includes monomeric meganucleases, dimeric meganucleases, and monomers that associate to form dimeric meganucleases.

In certain embodiments, the methods and compositions described herein utilize nucleases, including engineered (non-naturally occurring) homing endonucleases (meganucleases). I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I- Recognition sequences for homing endonucleases and meganucleases, such as TevII and I-TevIII, are known. US Pat. No. 5,420,032; US Pat. No. 6,833,252; Belfort et al. (1997) Nucleic Acids Res. Volume 25: 3379-3388; Dujon et al. (1989) Gene 82: 115-118; Perler et al. (1994) Nucleic Acids Res. Vol. 22, pp. 1125-1127; Jasin (1996) Trends Genet. Volume 12: pp. 224-228; Gimble et al. (1996) J. Mol. Mol. Biol. Volume 263: pp. 163-180; Argast et al. (1998) J. Mol. Mol. Biol. See also Volume 280: pp. 345-353 and the New England Biolabs Catalog. In addition, the DNA binding specificity of homing endonucleases and meganucleases can be engineered to bind to non-natural target sites. For example, Chevalier et al. (2002) Molec. Cell Vol. 10, pp. 895-905; Epinat et al. (2003) Nucleic Acids Res. 31: 2952-2962; Ashworth et al. (2006) Nature 441: 656-659; Paques et al. (2007) Currant Gene Therapy 7: 49-66; see U.S. Patent Application Publication No. 20070117128. I want to. The DNA-binding domains of homing endonucleases and meganucleases can be modified in the context of the nuclease as a whole (ie, such that the nuclease contains a homologous cleavage domain) or fused to a heterologous cleavage domain. Custom meganucleases are commercially available, for example, from New England Biolabs (Ipswich, MA) and can also customarily target and cleave any position in DNA.

Manipulation of a B cell can be performed, for example, by one or more vectors encoding a nuclease, such that a vector containing the encoded nuclease is incorporated by the B cell, such as one or more nucleases to the B cell (eg, one or more nucleases). , ZFN, TALEN, CRISPR / Cas, meganuclease). The vector can be a viral vector.

In some embodiments, the nuclease cleaves a specific endogenous locus (eg, a safe harbor gene or locus of interest) in a cell (eg, Memory B cell or plasma cell) and may be one or more. An extrinsic (donor) sequence (eg, a transgene) is administered (eg, one or more vectors containing these exogenous sequences). In such an embodiment, the donor sequence may encode a follistatin (eg, a follistatin transgene). The nuclease can induce double-strand breaks (DSBs) or single-strand breaks (nicks) in the target DNA. In some embodiments, targeted insertion of a donor transgene (eg, a follistatin donor transgene) is a homologous recombination repair (eg, foliastatin transgene) at the site of integration of the transgene into the genome of a cell (eg, a follistatin transgene). HDR), non-homologous repair mechanisms (eg, NHEJ-mediated terminal capture), or insertion and / or deletion of nucleotides (eg, endogenous sequences). In one embodiment, the method of transfecting B cells comprises electroporation of the B cells prior to contact of the vector with the B cells. In one embodiment, cells are electroporated on days ranging from day 1 to day 12 of in vitro culture. In one embodiment, cells are electroporated on days 1, 2, 3, 4, 5, 6, 7, 8 or 9 of in vitro culture. In one embodiment, cells are electroporated on day 2 of in vitro culture for delivery of the plasmid.

In one embodiment, cells are transfected using a transposon. As used herein, the term "transposonization" can refer to cells that, in some embodiments, are transfected with the transposon. A large number of transposon systems are known in the art and are suitable for use in the present invention. For example, sleeping beauty transposons and Piggybac transposons are well known in the art and are suitable for use in the present invention. For example, Hackett PB, et al., Evaluating Risks of Insertional Mutagenesis by DNA Transposons in Gene Therapy, Transl Res. 2013 April; 161 (4): 265, each of which incorporates them in their entirety herein by reference. -283; Hudecek M, et al., Going non-viral: the Sleeping Beauty transposon system breaks on through to the clinical side, Crit Rev Biochem Mol Biol. 2017 Aug; 52 (4): 355-380. In some embodiments, cells are transfected using a sleeping beauty transposon. The sleeping beauty transposon may be a T2 sleeping beauty transposon or a T4 sleeping beauty transposon in some embodiments. In some embodiments, the utilization of the sleeping beauty transposon system is to transfect B cells with a DNA construct encoding the mechanism of the transposon system and a DNA construct encoding a follistatin polypeptide (eg, electro). Can be included (by poration). In some embodiments, the DNA construct encoding the transposon mechanism may be pCMV-SB100x. In some embodiments, the use of a sleeping beauty transposon system involves transfecting B cells with a DNA construct encoding a follistatin polypeptide (eg, by electroporation), and further transposing B cells into a transposon system. Includes transfection with a mechanism-encoding mRNA. In some embodiments, the mRNA encoding the transposon system mechanism encodes SB100 × transposase. In some embodiments, cells are transfected using the Piggybac transposon. In one embodiment, cells are transfected using a transposon (eg, a T2 or T4 sleeping beauty transposon or Piggybac transposon) on days ranging from day 1 to day 12 of in vitro culture. In one embodiment, cells use a transposon (eg, T2 or T4 sleeping beauty transposon or Piggybac transposon) on days 1, 2, 3, 4, 5, 6, 7, 8, or 9 of in vitro culture. And transfect. In one embodiment, cells are transfected using a minicircle on days ranging from day 1 to day 12 of in vitro culture. In one embodiment, cells are transfected using a minicircle on days 1, 2, 3, 4, 5, 6, 7, 8, or 9 of in vitro culture.

In one embodiment, cells are transfected using a sleeping beauty transposon (eg, a T2 or T4 sleeping beauty transposon) on days ranging from day 1 to day 12 of in vitro culture. In one embodiment, cells are transduced by electroporation using a sleeping beauty transposon system (eg, T2 or T4 sleeping beauty transposon) on day 2 of in vitro culture. In one embodiment, cells are transduced by electroporation using a sleeping beauty transposon system (eg, T2 or T4 sleeping beauty transposon) on day 5 of in vitro culture. In one embodiment, cells are transduced by electroporation using a sleeping beauty transposon system (eg, T2 or T4 sleeping beauty transposon) on day 8 of in vitro culture. In one embodiment, cells are transduced by electroporation using a sleeping beauty transposon (eg, T2 or T4 sleeping beauty transposon) system on day 11 of in vitro culture. In one embodiment, cells are transduced by electroporation using a sleeping beauty transposon (eg, T2 or T4 sleeping beauty transposon) system on day 14 of in vitro culture.

In one embodiment, cells are transfected using the Piggybac transposon on days ranging from day 1 to day 12 of in vitro culture. In one embodiment, cells are transduced by electroporation using the Piggybac transposon on the second day of in vitro culture. In one embodiment, cells are transduced by electroporation using the Piggybac transposon on day 5 of in vitro culture. In one embodiment, cells are transduced by electroporation using the Piggybac transposon on day 8 of in vitro culture. In one embodiment, cells are transduced by electroporation using the Piggybac transposon on day 11 of in vitro culture. In one embodiment, cells are transduced by electroporation using the Piggybac transposon on day 14 of in vitro culture.

In one embodiment, B cells are contacted with a vector containing the nucleic acid of interest operably linked to a promoter under conditions sufficient to transfect at least a portion of the B cells. In one embodiment, B cells are contacted with a vector containing the nucleic acid of interest operably linked to a promoter under conditions sufficient to transfect at least 5% of the B cells. In a further embodiment, B cells are at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70 of B cells. The vector is contacted under conditions sufficient to transfect%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or even 100%. In one particular embodiment, B cells cultured in vitro are transfected as described herein, in which case the cultured B cells are at least 5%, 10% of the B cells. 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96% , 97%, 98%, 99% or even 100% are contacted with the vectors described herein under conditions sufficient to transfect.

Viral vectors can be used to transduce memory B cells and / or plasma cells. Examples of viral vectors include adenovirus-based vectors, adeno-associated virus (AAV) -based vectors, retroviral vectors, retroviral adenovirus vectors, and herpes simplex virus (HSV), including amplicon vectors, replication-deficient HSV and attenuated. Vectors derived from HSV are included without limitation (eg, Krisky, Gene Ther. 5, pp. 1517-30, 1998; Pfeifer, Annu. Rev., the entire of each of which is incorporated herein by reference. Genomics Hum. Genet. Vol. 2, pp. 177-211, 2001).

In one embodiment, cells are transduced with a viral vector (eg, lentiviral vector) on days 1, 2, 3, 4, 5, 6, 7, 8 or 9 of in vitro culture. In certain embodiments, cells are transduced with a viral vector on day 5 of in vitro culture. In one embodiment, the viral vector is a lentivirus. In one embodiment, cells are transduced with measles virus pseudotyped lentivirus on day 1 of in vitro culture.

In one embodiment, B cells are transduced with a retroviral vector using any of a variety of known techniques in the art (eg, Science, April 12, 1996, Vol. 272: 263-. 267 pages; Blood 2007, 99: 2342-2350; Blood 2009, 113: 1422-1431; Blood October 8, 2009; 114 (15): 3173-80; Blood 2003; Vol. 101 (No. 6): pp. 2167-2174; See Currant Protocols in Molecular Biology or Current Protocols in Immunology, John Willey & Sons, New York, NY (200). Additional statements regarding viral transduction of B cells can be found in WO2011 / 0852447 and WO2014 / 152832, each of which is incorporated herein by reference in its entirety.

For example, donor-derived PBMC, B or T lymphocytes, and other B cell cancer cells such as B-CLL have been isolated and described in IMDM medium or RPMI 1640 (GibcoBRL Invitrogen, Attackland, New Zealand), or herein Serum-free or in other suitable media described, such as serum (eg, 5-10% FCS, human AB serum and serum substitutes) and penicillin / streptomycin, and / or transferrin and / or insulin. Suitable supplements can be supplemented and cultured. In one embodiment, the cells 48 were seeded at 1 × 10 ⁵ cells in the well plates, the concentrated vectors at various doses may be routinely optimized by those skilled in the art using conventional methodology Is added. In one embodiment, B cells are transferred to the MS5 cell monolayer in RPMI supplemented with 10% AB serum, 5% FCS, 50 ng / ml rhSCF, 10 ng / ml rhhlL-15 and 5 ng / ml rhhlL-2 and medium. Is regularly renewed as needed. Other suitable media and supplements, as will be recognized by those of skill in the art, can be used as desired.

Certain embodiments relate to the use of retroviral vectors, or vectors derived from retroviruses. An envelope that can infect animal cells and utilizes reverse transcriptase in the early stages of infection to produce a DNA copy from its RNA genome, which is then typically integrated into the host genome. It is a type RNA virus. Examples of retroviral vectors include moloney murine leukemia virus (MLV) -derived vectors, mouse stem cell virus-based retroviral vectors that provide long-term stable expression in target cells such as hematopoietic precursor cells and their differentiated progeny (eg, Howley et al.). , PNAS USA, Vol. 93: 10297-10302, 1996; Keller et al., Blood 92: 877-887, see 1998), Hybrid Vectors (eg, Choi et al., Stem Cells, Vol. 19, pp. 236-246, (See 2001), as well as complex retroviral-derived vectors such as the lentiviral vector.

In one embodiment, B cells are contacted with a retroviral vector containing the nucleic acid of interest operably linked to a promoter under conditions sufficient to transduce at least a portion of the B cells. In one embodiment, B cells are contacted with a retroviral vector containing the nucleic acid of interest operably linked to a promoter under conditions sufficient to transduce at least 2% of the B cells. In a further embodiment, B cells are at least 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% of resting B cells. Enough to transduce 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or even 100% Contact with the vector under various conditions. In one particular embodiment, differentiated and activated B cells cultured in vitro as described herein are transduced, in this case the cultured differentiated / activated B cells. Cells are at least 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50 of differentiated and activated B cells. Sufficient to transduce%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or even 100% Under conditions, it is contacted with the vectors described herein.

In certain embodiments, prior to transduction, cells are subjected to appropriate concentrations of Staphylococcus aureus Cowan (SAC; Calbiochem, San Diego, CA) and / or IL- which are known to those of skill in the art and are customarily optimized. Pre-stimulated with 2. Other B cell activating factors (eg, PMA) can be used as known to those of skill in the art and described herein.

As noted above, certain embodiments use lentiviral vectors. The term "lentivirus" refers to a genus of complex retroviruses that can infect both dividing and non-dividing cells. Examples of lentiviruses include HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2), Bisna maedi, goat arthritis / encephalitis virus, horse infectious anemia virus, feline immunodeficiency virus (FIV), bovine immunodeficiency. Deficiency virus (BIV) and simian immunodeficiency virus (SIV). The lentiviral vector can be derived from any one or more of these lentiviruses (eg, Evans et al., Hum Gene Ther. 10: Incorporating all of these by reference herein: 1479 to 1489, 1999; Case et al., PNAS USA 96: 2988 to 2939, 1999; Uchida et al., PNAS USA 95: 1 1939 to 1 1944, 1998; Miyoshi et al., Science 283: 682. ~ 686, 1999; Sutton et al., J Viral 72: 5781-5788, 1998; and Frecha et al., Blood. 1 12: 4843-52, 2008).

It has been recorded that resting T and B cells can be transduced by VSVG-coated LV carrying most of the HIV accessory proteins (vif, vpr, vpu and nef) (eg, Frecha et al., 2010, Mol. Therapy Vol. 18, p. 1748). In certain embodiments, the retroviral vector comprises a particular minimal sequence from a lentiviral genome, such as the HIV or SIV genome. The lentivirus genome typically has a 5'end repeat (LTR) region, a gag gene, a pol gene, an env gene, an accessory gene (eg, nef, viv, vpr, vpu, tat, rev) and a 3'LTR region. Is organized into. The viral LTR is divided into three regions called U3, R (repetitive) and U5. The U3 region contains enhancer and promoter elements, the U5 region contains a polyadenylation signal, and the R region separates the U3 and U5 regions. The transcribed sequence of the R region appears at both the 5'and 3'ends of the viral RNA (eg, RNA Viruses: A Practical Application, which incorporates all of these by reference herein) (Alan J. et al. Cann ed., Oxford University Press, 2000); O Narayan, J. Gen. Virus. 70: 1617-1639, 1989; Fields et al., Fundamental Virology Virus., 1990; Volume: 8150-7, 1998; and US Pat. No. 6,013,516). A lentiviral vector comprises one or more of these elements of the lentiviral genome and can regulate the activity of the vector as desired, or is deficient in one or more of these elements. Loss, insertion, substitution or mutation can be included to reduce, for example, the pathological effect of lentiviral replication, or the lentiviral vector can be limited to a single round of infection.

Typically, the smallest retroviral vector is a particular 5'LTR and 3'LTR sequence, one or more genes of interest (which should be expressed in a target cell), one or more promoters, and Contains cis-acting sequences for RNA packaging. Other regulatory sequences may be included as described herein and known in the art. Viral vectors are typically cloned into plasmids that can be transfected into packaging cell lines such as eukaryotic cells (eg, 293-HEK) and typically contain sequences useful for plasmid replication in bacteria. ..

In certain embodiments, the viral vector comprises a sequence from a retrovirus 5'and / or 3'LTR, such as a lentivirus. The LTR sequence can be an LTR sequence derived from any lentivirus from any species. For example, the LTR sequence can be an LTR sequence derived from HIV, SIV, FIV or BIV. Preferably, the LTR sequence is an HIV LTR sequence.

In certain embodiments, the viral vector comprises the Lentiviral 5'LTR-derived R and U5 sequences, as well as the lentiviral-derived inactivated or "self-inactivated" 3'LTR. A "self-inactivating 3'LTR" is a 3'end repeat sequence (LTR) containing a mutation, substitution or deletion that prevents the LTR sequence from driving the expression of a downstream gene. A copy of the U3 region from the 3'LTR acts as a template for the production of both LTRs in the integrated provirus. Therefore, if a 3'LTR with an inactivated deletion or mutation is incorporated as a provirus 5'LTR, transcription from the 5'LTR is not possible. This eliminates competition between the virus enhancer / promoter and any internal enhancer / promoter. Self-inactivating 3'LTRs are incorporated herein by reference in their entirety, for example, by Zuffery et al., J Virol. Volume 72: pp. 9873-9880, 1998; Miyoshi et al., J Virol. 72: 8150-8157, 1998; and Iwakuma et al., J Virology 261: 120-132, 1999. The self-inactivating 3'LTR can be produced by any method known in the art. In certain embodiments, the U3 element of the 3'LTR has a deletion in its enhancer sequence, preferably the TATA box, Spl and / or NF-copper B site. As a result of the self-inactivated 3'LTR, the provirus integrated into the host cell genome will include an inactivated 5'LTR.

The vectors provided herein typically include a gene encoding a protein that is preferably expressed in one or more target cells, such as follistatin. However, the vectors provided herein also include genes encoding other molecules that are preferably expressed in one or more target cells (eg, siRNA). In some embodiments, in the viral vector, the gene of interest (eg, follistatin) is preferably located between the 5'LTR and 3'LTR sequences. Moreover, in some embodiments, the gene of interest (eg, follistatin) is preferably in a functional relationship with a transcriptional regulatory sequence, such as another genetic element, such as a promoter and / or enhancer. Once the gene is taken up by the target cell, it regulates the expression of the gene of interest (eg, follistatin) in a particular manner. In certain embodiments, useful transcriptional regulatory sequences are sequences that are highly regulated in terms of activity, both temporally and spatially.

In certain embodiments, one or more additional genes are incorporated as a safety measure to selectively kill or deplete transfected target cells within a heterogeneous population, eg, within a human patient. Can be made possible. In one non-limiting exemplary embodiment, the gene is a thymidine kinase gene (TK) whose expression makes target cells sensitive to the action of the drug ganciclovir. In some embodiments, the additional gene is a tag on the cell surface protein. In some embodiments, the gene is a suicide gene. In some embodiments, the suicide gene is a caspase-9 suicide gene activated by a dimerization agent (see, eg, Tey et al., Biology of Blood and Marrow Transplantation 13: 913-924, 2007). I want to be).

In certain embodiments, one or more additional genes encoding the marker protein are placed before or after the primary gene (eg, the follistatin gene) in a viral or non-viral vector to produce the desired protein. It can enable the identification and / or selection of cells expressing (eg, follistatin). Certain embodiments incorporate additional cell surface proteins that can facilitate the identification and / or selection of cells expressing the desired protein (eg, follistatin). Certain embodiments incorporate fluorescent marker proteins, such as green fluorescent protein (GFP) or red fluorescent protein (RFP), along with the primary gene of interest (eg, the follistatin gene). An IRES sequence or 2A element that isolates the primary gene of interest (eg, the follistatin gene) from the reporter gene and / or any other gene of interest if one or more additional reporter genes are included. May also be included.

Certain embodiments can use genes encoding one or more selectable markers. As an example, selectable markers that are effective in eukaryotic or prokaryotic cells, such as genes for drug resistance encoding factors required for survival or growth of transformed host cells grown in selective culture medium. Can be mentioned. An exemplary selection gene confer resistance to antibiotics or other toxins such as G418, hygromycin B, puromycin, zeocin, vavine, blastsidedin, ampicillin, neomycin, methotrexate or tetracycline. It encodes proteins that complement or supplement auxotrophic deficiencies, which are present on separate plasmids and can be introduced by cotransfection with viral vectors. In one embodiment, the gene encodes a mutant dihydrofolate reductase (DHFR) that confers methotrexate resistance. Certain other embodiments include one or more cell surface receptors that can be used for tagging and detection or purification of transfected cells (eg, low affinity nerve growth factor receptor (LNGFR)). )), Or genes encoding other such receptors useful as transfection tag systems can be used. For example, Lauer et al., Cancer Gene Ther. March 2000; see Volume 7 (No. 3): pp. 430-7.

Certain viral vectors, such as retroviral vectors, use one or more heterologous promoters, enhancers, or both. In certain embodiments, the U3 sequence from the retrovirus or lentivirus 5'LTR can replace the promoter or enhancer sequence in the virus construct. Certain embodiments use "internal" promoters / enhancers that are located between the 5'LTR and 3'LTR sequences of the viral vector and operably linked to the gene of interest (eg, the follistatin gene).

"Functional relationships" and "operably linked" indicate that when a promoter and / or enhancer is contacted with the appropriate regulatory molecule, the expression of the gene (eg, the follistatin gene) is affected. As such, it means without limitation that the gene (eg, the follistatin gene) is in the correct position and orientation with respect to the promoter and / or enhancer. Any enhancer / promoter that regulates (eg, increases, decreases) expression of the viral RNA genome in packaging cell lines, regulates expression of selected genes of interest in infected target cells, or both. Combinations of are also available.

A promoter is an expression control element formed by a DNA sequence that allows polymerase binding and transcription to occur. The promoter is located upstream (5') of the start codon of the selected gene of interest (typically within about 100-1000 bp) and is operably linked to the transcription and translation of the coding polynucleotide sequence. An untranslated sequence that controls. Promoters can be inducible or constitutive. Under its control, the inducible promoter initiates increased levels of transcription from DNA in response to any changes in culture conditions, such as temperature changes. Promoters can be unidirectional or bidirectional. Bidirectional promoters can be used to co-express two genes, eg, the gene of interest, such as follistatin, and a selectable marker. Alternatively, bidirectional promoter configurations can be utilized that include two promoters that control the expression of different genes in the same vector with opposite orientations.

Various promoters are known in the art as well as methods for operably linking promoters to polynucleotide coding sequences. Both the native promoter sequence and many heterologous promoters can be used to direct the expression of the selected gene of interest. Certain embodiments generally use heterologous promoters as they allow better transcription and higher yields of the desired protein as compared to native promoters.

Certain embodiments can use heterologous viral promoters. Examples of such promoters are from viral genomes such as polyomavirus, poultry virus, adenovirus, bovine papillomavirus, trisarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and monkeyvirus 40 (SV40). Examples include the resulting promoter. Certain embodiments can use heterologous mammalian promoters, such as actin promoters, immunoglobulin promoters, heat shock promoters, or promoters associated with the native sequence of the gene of interest (eg, the follistatin gene). Typically, the promoter is compatible with the target cell, such as activated B lymphocytes, trait B cells, memory B cells or other lymphocyte target cells.

Certain embodiments may use one or more of the RNA polymerase II and III promoters. Suitable selections of the RNA polymerase III promoter are described, for example, in Paule and White. , Nucleic Acids Research. , 28, 1283-1298, 2000. The RNA polymerase II and III promoters also include either synthetic or engineered DNA fragments that can orient the RNA polymerase II or III to transcribe its downstream RNA coding sequence, respectively. In addition, the RNA polymerase II or III (Pol II or III) promoter (s) used as part of the viral vector can be inducible. Either suitable inducible Pol II or III promoter can be used with the methods described herein. Exemplary Pol II or III promoters are incorporated herein by reference in their entirety, Ohkawa and Taira, Human Gene Therapy, Vol. 11, pp. 577-585, 2000; and Meissner et al., Nuclear Acids Research, et al. Includes the Tetracycline Responsive Promoter, Vol. 29, pp. 1672–1682, provided in 2001.

Non-limiting examples of constitutive promoters that can be used include ubiquitin promoters, CMV promoters (see, eg, Karasuyama et al., J. Exp. Med. 169: p. 13, 1989), β-actin (eg, eg. Gunning et al., PNAS USA Vol. 84: 4831-4835, 1987), Elongation Factor-1 Alpha (EF-1 Alpha) Promoter, CAG Promoter and pgk Promoter (see, eg, each of these herein by reference). Incorporates, Adra et al., Gene 60: 65-74, 1987); Singer-Sam et al., Gene 32: 409-417, 1984; and Dobson et al., Nuclear Acids Res. Volume 10: pp. 2635-2637, 1982). As non-limiting examples of tissue-specific promoters, the lck promoter (eg, Garvin et al., Mol. Cell Biol. 8: 3058-3064, 1988; and Takadera et al., Mol. Cell Biol. 9: 2173-2180. , 1989), Myogenin promoter (Yee et al., Genes and Development 7: 1277-1289, 1993) and three promoter (eg, Gundersen et al., Gene 11 13: 207-214, 1992). See).

Additional examples of promoters are the ubiquitin-C promoter, the human μ heavy chain promoter or the Ig heavy chain promoter (eg, MH), and the human kappa light chain promoter or the Ig light chain promoter (eg, MH), which are functional in B lymphocytes. For example, EEK) can be mentioned. The MH promoter contains a human μ heavy chain promoter preceded by an iEμ enhancer adjacent to the matrix association region, and the EEK promoter is preceded by an intron enhancer (iEκ), a matrix association region and a 3'enhancer (3Eκ). It contains a κ light chain promoter (see, eg, Luo et al., Blood. 1, Volume 13, pp. 1422-1431, 2009 and US Patent Application Publication No. 2010/0203630). Thus, certain embodiments may use one or more of these promoter or enhancer elements.

In one embodiment, one promoter drives the expression of selectable markers and the second promoter drives the expression of the gene of interest (eg, the follistatin gene). For example, in one embodiment, the EF-1 alpha promoter drives the production of selectable markers (eg, DHFR) and the miniature CAG promoter (eg, Fan et al., Human Gene Therapy, Vol. 10, pp. 2273-2285, 1999). (See) drives the expression of the gene of interest (eg, follistatin).

As noted above, certain embodiments use enhancer elements, such as internal enhancers, to increase the expression of the gene of interest. Enhancers are cis-acting elements of DNA, usually about 10-300 bp in length, that act on promoters to increase their transcription. The enhancer sequence can be derived from mammalian genes such as ιεμ enhancer, ιεκ intron enhancer and 3'εκ enhancer (eg, globin, elastase, albumin, α-fetoprotein, insulin). Also included are eukaryotic virus-derived enhancers, including SV40 enhancers (bp 100-270) posterior to the origin of replication, cytomegalovirus early promoter enhancers, polyoma enhancers posterior to the origin of replication, and adenovirus enhancers. The enhancer can be spliced into the vector at position 5'or 3'with respect to the antigen-specific polynucleotide sequence, but is preferably located 5'from the promoter. One of ordinary skill in the art will select an appropriate enhancer based on the desired expression pattern.

In certain embodiments, the promoter is selected to allow inducible expression of the gene of interest (eg, the follistatin gene). Numerous systems for inducible expression are known in the art, including tetracycline responsive systems and lac operator-repressor systems. It is also contemplated that a combination of promoters can be used to obtain the desired expression of the gene of interest (eg, the follistatin gene). One of ordinary skill in the art will be able to select a promoter based on the desired expression pattern of the gene in the organism of interest and / or the target cell.

Certain viral vectors contain cis-acting packaging sequences to facilitate the uptake of genomic viral RNA into viral particles. An example is the psi-sequence. Such cis-acting sequences are known in the art. In certain embodiments, the viral vectors described herein can express two or more genes, eg, an internal ribosome entry sequence. Separate across the first gene by incorporating elements that facilitate co-expression, such as the (IRES) element (US Pat. No. 4,937,190 incorporated herein by reference) and / or the 2A element. It can be achieved by incorporating an internal promoter operably linked to each of the genes of. As a mere explanation, an IRES or 2A element can be used if a single vector contains a sequence encoding each strand of an immunoglobulin molecule with the desired specificity. For example, the first coding region (coding either the heavy chain or the light chain) can be located just downstream of the promoter, and the second coding region (coding the other chain) is the first coding. It can be located downstream from the region and the IRES or 2A element is located between the first and second coding regions, preferably directly in front of the second coding region. In other embodiments, the IRES or 2A element is used for co-expression of unrelated genes, such as reporter genes, selectable markers, cell surface proteins, or genes that enhance immune function. Examples of IRES sequences that can be used are encephalomyelitis virus (EMCV), mouth-foot epidemic virus (FMDV), Tyler mouse encephalomyelitis virus (TMEV), hitrinovirus (HRV), coxsackie virus (CSV), poliovirus ( POLIO), hepatitis A virus (HAV), hepatitis C virus (HCV) and pestivirus (eg, porcine cholera virus (HOCV) and bovine viral diarrhea virus (BVDV)) include without limitation the IRES elements (eg). Le et al., Virus Genes Vol. 12, pp. 135-147, 1996; and Le et al., Nuc. Acids Res. Vol. 25: pp. 362-369, 1997. reference). An example of a 2A element is an F2A sequence derived from a foot-and-mouth disease virus.

In certain embodiments, the vectors provided herein also contain additional genetic elements to achieve the desired result. For example, certain viral vectors can include signals that facilitate nuclear invasion of the viral genome in target cells, such as the HIV-1 flap signal. As yet another example, certain viral vectors can include elements that facilitate the characterization of provirus integration sites in target cells, such as tRNA amber suppressor sequences. Certain viral vectors can contain one or more genetic elements designed to enhance the expression of the gene of interest (eg, the follistatin gene). For example, woodchuck hepatitis virus responsive elements (WREs) can be placed in the construct (eg, all of these incorporated herein by reference, Zuffery et al., J. Virus. 74: 3668-3681. P. 1999; and Deglon et al., Hum. Gene Ther. 11, pp. 179-190, 2000). As another example, a chicken β-globin insulator may be included in the construct. This element has been shown to reduce the probability of silencing of integrated DNA in target cells due to methylation and heterochromatinization effects. In addition, the insulator can shield internal enhancers, promoters and extrinsic genes from positive or negative positional effects from surrounding DNA at the integration site on the chromosome. Certain embodiments use each of these genetic elements. In another embodiment, the viral vectors provided herein can also contain a ubiquitous chromatin opening element (UCOE) for increased expression (eg, Zhang F et al., Molecular Therapy: The). journal of the American Society of Gene Therapy September 2010; see Vol. 18 (No. 9), pp. 1640-9).

In certain embodiments, the viral vectors provided herein (eg, retroviruses, lentiviruses) are primarily selected from one or more species to target selected cell types. It is "pseudotyped" by the viral glycoprotein or enveloped protein. Pseudotyping generally refers to the uptake of one or more heterologous viral glycoproteins onto cell surface viral particles, which often select the viral particles differently from their normal target cells. Can infect cells. The "heterologous" element is derived from a virus other than the virus from which the RNA genome of the viral vector is derived. Typically, the glycoprotein coding region of a viral vector is genetically altered, such as by a deletion to prevent the expression of its own glycoprotein. Simply as an explanation, the envelope glycoproteins gp41 and / or gp120 from HIV-derived lentiviral vectors are typically deleted prior to pseudotyping with heterologous viral glycoproteins.

In certain embodiments, the viral vector is pseudotyped by a heterologous viral glycoprotein that targets B lymphocytes. In certain embodiments, viral glycoproteins allow for selective infection or transduction of resting or resting B lymphocytes. In certain embodiments, viral glycoproteins allow selective infection of B lymphocyte plasma cells, plasmablasts and activated B cells. In certain embodiments, viral glycoproteins allow infection or transduction of quiescent B lymphocytes, plasmablasts, plasma cells and activated B cells. In certain embodiments, viral glycoproteins allow infection of B-cell chronic lymphocytic leukemia cells. In one embodiment, the viral vector is pseudotyped by VSV-G. In another embodiment, the heterologous viral glycoprotein is derived from a measles virus glycoprotein such as Edmonton measles virus. Certain embodiments pseudotype the measles virus glycoprotein hemagglutinin (H), fusion protein (F), or both (eg, all of these incorporated herein by reference, Frecha et al., Blood. 1 12: 4843-52, 2008; and Frecha et al., Blood. 1 14: 3173-80, 2009). In one embodiment, the viral vector is pseudotyped with the Gibbon ape leukemia virus (GALV). In one embodiment, the viral vector is pseudotyped by a cat endogenous retrovirus (RD114). In one embodiment, the viral vector is pseudotyped by a baboon endogenous retrovirus (BaEV). In one embodiment, the viral vector is pseudotyped by the murine leukemia virus (MLV). In one embodiment, the viral vector is pseudotyped with the Gibbon ape leukemia virus (GALV). In a further embodiment, the viral vector has impacted antibody binding, such as one or more variable regions (eg, heavy and light chain variable regions) that function to target the vector to a particular cell type. Includes domain.

Viral vectors have been generated, for example, by Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY (1989)), Coffin et al. (RetrovirusN. 1997)) and "RNA Viruses: A Plastic Applications" (edited by Alan J. Cann, Oxford University Press (2000)), restricted endonuclease digestion, ligation, transformation, plasmid purification, PCR amplification. And can be achieved using any suitable genetic manipulation technique known in the art, including without limitation standard techniques for DNA sequencing.

Any of a variety of methods known in the art can be used to produce suitable retroviral particles whose genome contains an RNA copy of a viral vector. As one method, the viral vector can be introduced into a packaging cell line that packages the viral genomic RNA based on the viral vector to the viral particles with the desired target cell specificity. Packaging cell lines, typically trans, provide the viral proteins required for packaging viral genomic RNA into viral particles and infecting target cells, including structural gag proteins, enzyme pol proteins and enveloped glycoproteins.

In certain embodiments, the packaging cell line stably expresses certain required or desired viral proteins (eg, gag, pol) (eg, incorporated herein by reference, U.S. Pat. No. 6,037,045. , 218, 181). In certain embodiments, the packaging cell line comprises a particular particular viral protein (eg, gag, pol, glycoprotein) of need or desire, comprising the measles viral glycoprotein sequences described herein. The plasmid that encodes the one is transiently transfected. In one exemplary embodiment, the packaging cell line stably expresses the gag and pol sequences, which in turn is transfected with a plasmid encoding a viral vector and a plasmid encoding a glycoprotein. After introduction of the desired plasmid, the virus particles are harvested and processed accordingly, such as by ultracentrifugation, to achieve a concentrated stock of virus particles. Exemplary packaging cell lines are 293 (ATCC CCL X), HeLa (ATCC CCL 2), D17 (ATCC CCL 183), MDCK (ATCC CCL 34), BHK (ATCC CCL-10) and Cf2Th (ATCC CRL 1430). ) Includes cell lines.

Therapeutic Agent In the present specification, the "gene of interest" or "gene" or "nucleic acid of interest" refers to a transgene to be expressed in a target transfected cell. In certain embodiments, the gene is a follistatin gene. Although the term "gene" can be used, the term does not imply that it is a gene found in genomic DNA and is used interchangeably with the term "nucleic acid". In general, the nucleic acid of interest provides a nucleic acid suitable for the coding of a therapeutic agent (eg, follistatin) and can contain cDNA or DNA, which may or may not contain introns, but generally contains introns. Absent. As noted elsewhere, the nucleic acid of interest is operably linked to an expression control sequence for effective expression of the protein of interest in the target cell. In certain embodiments, the vectors described herein can contain one or more genes of interest, eg, tissue using the internal promoters described herein. It can contain 2, 3, 4 or 5 or more genes of interest, such as heavy and light chains of immunoglobulins that can be converted.

References to "polynucleotide" or "nucleic acid" specify mRNA, RNA, cRNA, cDNA or DNA herein. The term typically refers to a polymeric form of a nucleotide that is at least 10 bases long, which is a modified form of a ribonucleotide or deoxynucleotide, or either type of nucleotide. The term includes single- and double-stranded forms of DNA and RNA. The nucleic acid or gene of interest can be any nucleic acid encoding the protein of interest.

In some embodiments, any one of the embodiments disclosed herein can utilize the gene of interest which is a follistatin protein. The follistatin protein may be any one of the follistatin proteins set forth in SEQ ID NOs: 1 to 4 in some embodiments. Thus, in some embodiments, the therapeutic agent delivered to the genetically modified B cells may be a follistatin protein, as described herein.

Follistatin In various aspects, the present disclosure relates to B cells engineered to express a follistatin (eg, one or more follistatin peptides). As used herein, the term "follistatin" refers to a follistatin-related protein derived from a family of follistatin (FST) proteins and any species. Follistatin is an autocrine glycoprotein expressed in almost all tissues of higher animals. Follistatin was first isolated from follicular fluid and identified as a protein fraction that inhibits the secretion of follicle-stimulating hormone (FSH) from the anterior pituitary gland and was therefore designed as an FSH inhibitory protein (FSP). Its major function was then determined to be, for example, activin, the binding and neutralization of members of the TGF-β superfamily, including the paracrine hormone that enhances FSH secretion in the anterior pituitary gland.

In some embodiments, the term "follistatin polypeptide" or "follistatin" refers to any naturally occurring polypeptide of the follistatin family and, for example, ligand binding (eg, myostatin, GDF-11, activin A, etc.). It is used to refer to polypeptides containing any variant (variant, fragment, fusion, and peptide mimicking form) thereof that retains useful activity, including activin B) or heparin binding. For example, in some embodiments, the follistatin polypeptide has a sequence that is at least about 80% identical to the sequence of the follistatin polypeptide, preferably at least 85%, 90%, 95%, 97%, 99. Percentages or higher may include polypeptides comprising an amino acid sequence derived from any known follistatin sequence having an identity of% or higher.

Follistatin is a single chain polypeptide having a molecular weight range of 31-49 kDa based on alternative splicing of mRNA and variable glycosylation of proteins. The human gene encoding follistatin (FST) has six exons on chromosome 5q11.2 over 5329 bp, giving rise to two major transcripts: transcript variant FST344 (1122 bp) and transcript FST317 (1386 bp). .. Exon 1 in FST encodes the follistatin signal peptide, exon 2 encodes the follistatin N-terminal domain, and each of exons 3-5 encodes the follistatin module. By alternative splicing, either one of exon 6A (encoding the acidic region in FST344) or exon 6B (containing two bases of the stop codon of FST317) is utilized (Shimasaki, S. et al., 1988). ).

After removal of the 29 amino acid signal peptides by such selectively spliced mRNAs (FST344 and FST317), two amino acids, 315 amino acids (ie, FST315) and 288 amino acids (ie, FST288), respectively. A follistatin protein is produced, which can be further degraded to follistatin 303 (FST303) by proteolysis. Analysis of the amino acid sequence revealed that the native human follistatin polypeptide had five domains (from the N-terminal side): a signal sequence peptide (amino acids 1-29 of SEQ ID NO: 1), an N-terminal domain (FSN) (of SEQ ID NO: 1). Amino acids 30-94), follistatin domain I (FSDI) (amino acids 95-164 of SEQ ID NO: 1), follistatin domain II (FSDII) (amino acids 168-239 of SEQ ID NO: 1), and follistatin domain III (FSDIII). It was found to contain (amino acid 245-316 of SEQ ID NO: 1). See PNAS, U.S.A., 1988, Vol. 85, No 12, pp 4218-4222.

The human follistatin-288 (FST288) precursor (ie, FST317) has the following amino acid sequence, the signal peptide is shown in bold, the N-terminal domain (FSN) is underlined, and the follistatin domains I- III (FSI, FSII, FSIII) is double underlined.

The processed (mature) human follistatin variant (FST288) has the following amino acid sequence, with the N-terminal domain underlined and follistatin domains I-III underlined. In addition, either the first amino acid G or N prior to the first cysteine can be removed or deliberately eliminated by processing, regardless of the outcome, such a slightly smaller poly. It is recognized that the polypeptide containing the peptide is further included.

The human follistatin-315 (FST315) precursor (ie, FST344) has the following amino acid sequence, the signal peptide is shown in bold, the N-terminal domain (FSN) is underlined, and the follistatin domains I-III (FSI, FSII, FSIII) are double underlined (NCBI Accession No. AAH04107.1; 344 amino acids).

Processed (mature) human FST315 has the following amino acid sequence, the N-terminal domain is single underlined and the follistatin domains I-III are double underlined. In addition, either the first amino acid G or N prior to the first cysteine can be removed or deliberately eliminated by processing, regardless of the outcome, such a slightly smaller poly. It is recognized that the polypeptide containing the peptide is further included.

The follistatin polypeptides of the present disclosure may include any naturally occurring domain of the follistatin protein and its variants (eg, variants, fragments, and peptide mimetic forms) that retain useful activity. For example, FST315 and FST288 have high affinities for both activin (activin A and activin B) and myostatin (and the closely related GDF11), and the follistatin domains (eg, FSN and FSDI-III) have such an affinity. It is well known that it is thought to be involved in the binding of such TGF-β ligand. However, it is believed that each of these three domains may have different affinities for such TGF-β ligands. For example, one study demonstrated that a polypeptide construct containing only one N-terminal domain (FSN) and two FSDI domains in tandem retains a high affinity for myostatin and was introduced into mice by gene expression. In some cases, it was demonstrated that it promoted systemic muscle growth with little or no affinity for activin (Nakatani et al., The FASEB Journal, Vol. 22477-487 (2008)). ).

Therefore, the present disclosure partially selects a given TGF-β ligand for a naturally occurring FST protein, eg, maintaining a high affinity for myostatin while significantly reducing its affinity for activin. Includes variant follistatin proteins that demonstrate binding and / or inhibition.

Thus, this disclosure is a polynucleotide (isolated, purified or pure polynucleotide) encoding a therapeutic agent of the present disclosure (eg, foristatin) for genetically modifying B cells, such a polynucleotide. Vectors comprising (including cloning and expression vectors), and cells transformed or transfected with a polynucleotide or vector (eg, host cells) according to the present disclosure. In certain embodiments, any one of the embodiments disclosed in the present disclosure is a follistatin selected from the follistatin polypeptides of SEQ ID NOs: 1-4 (eg, for expression in B cells). Can be used. In certain embodiments, any one of the embodiments disclosed in the present disclosure may utilize the human follistatin FST344 splice site variant, follistatin (eg, for expression in B cells). In certain embodiments, polynucleotides (DNA or RNA) encoding the protein of interest of the present disclosure (eg, follistatin) are contemplated. An expression cassette encoding a protein of interest is also contemplated in the present invention.

The present disclosure also relates to vectors containing the polynucleotides of the present disclosure, especially to recombinant expression constructs. In one embodiment, the disclosure comprises a polynucleotide encoding a protein of the present disclosure (eg, follistatin), along with other polynucleotide sequences that induce or facilitate transcription, translation and processing of such protein coding sequences. Intended a vector. Suitable cloning and expression vectors for use by prokaryotic and eukaryotic hosts are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, NY (1989). An exemplary cloning / expression vector is a plasmid known in the art, a phagemid, a phagemid, a cosmid, a virus, an artificial chromosome, suitable for amplification, transfer and / or expression of the polynucleotide contained therein. Includes cloning vectors, shuttle vectors and expression constructs that can be based on a chromosome or any nucleic acid vehicle.

As used herein, unless otherwise stated with respect to a viral vector, "vector" means a nucleic acid molecule capable of transporting another nucleic acid linked to it. Exemplary vectors include plasmids, minicircles, transposons (eg, sleeping beauty transposons), yeast artificial chromosomes, self-replicating RNA and viral genomes. One particular vector can autonomously replicate in the host cell, while another vector integrates into the host cell's genome, which allows it to replicate with the host genome. In addition, certain vectors contain, as used herein, nucleic acid sequences operably linked to expression control sequences (thus, expression control sequences can direct expression of such sequences). , "Recombinant expression vector" (or simply "expression vector"). In certain embodiments, the expression construct is derived from a plasmid vector. The construct for explanation is a modified pNASS vector (Clontech, Palo Alto, CA) having a nucleic acid sequence encoding an ampicillin resistance gene, a polyadenylation signal and a T7 promoter site; pDEF38 and pNEF38 (CMC ICS Biologies) with the CHEF1 promoter. Inc.); as well as pD18 (Lonza) with a CMV promoter. Other suitable mammalian expression vectors are well known (eg, Ausube et al., 1995; Sambrook et al., See above; eg, Invitrogen, San Diego, CA; Novagen, Madison, Wl; Pharmacia, Piscataway, NJ. See also the catalog).

Useful constructs containing the dihydrofolate reductase (DHFR) coding sequence can be prepared under regulatory control suitable for promoting enhanced production levels of the fusion protein, such levels being the appropriate drug of choice (eg, for example. , Methotrexate) due to gene amplification after application. In one embodiment, the use of a bifunctional transposon encoding a therapeutic gene (eg, FST) with drug-resistant DHFR in combination with incubation in methotrexate (MTX) to concentrate B cells that have been successfully translocated Produces stronger products.

In general, recombinant expression vectors are derived from origins of replication and selectable markers that allow transformation of host cells, as well as promoters derived from highly expressed genes to direct transcription of downstream structural sequences, as described above. Will include. Vectors operably linked to the polynucleotides according to the present disclosure yield cloning or expression constructs. An exemplary cloning / expression construct contains at least one expression control element, eg, a promoter, operably linked to the polynucleotides of the present disclosure. Additional expression control elements, such as enhancers, factor-specific binding sites, terminators and ribosome binding sites, are also contemplated in the vectors and cloning / expression constructs according to the present disclosure. The heterologous structural sequences of the polynucleotides according to the present disclosure are assembled in appropriate phases with the translation start and end sequences. Thus, for example, the coding nucleic acid provided herein can be any one of a variety of expression vector constructs (eg, minicircles) as a recombinant expression construct for expressing such a protein in a host cell. May be included.

Suitable DNA sequences (s) can be inserted into the vector by various procedures, for example. Generally, the DNA sequence is inserted into the appropriate restriction endonuclease cleavage site (s) by procedures known in the art. Standard techniques for cloning, DNA isolation, amplification and purification, as well as various isolation techniques, have been contemplated for enzymatic reactions involving DNA ligases, DNA polymerases, restriction endonucleases and others. A number of standard techniques have been developed, for example, by Ausubel et al. (Cold Spring Harbor Laboratory, Greene Publ. Assoc. Inc. & John Willy & Sons, Inc., Boston, MA, 1993). , Cold Spring Harbor Laboratory, Planview, NY, 1989); Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, NY, 1982, Volume II, Volume II, Volume II, Volume II, Volume II, Volume II, Volume II, Volume II, , UK, 1985); Hames and Higgins (eds.) (Molecular Acid Biology, IRL Press, Oxford, UK, 1985); and others.

The DNA sequence in the expression vector is operably linked to at least one suitable expression control sequence (eg, a constitutive promoter or a regulatory promoter) to direct mRNA synthesis. As described above, a typical example of such an expression control sequence is a promoter of a eukaryotic cell or a virus thereof. The promoter region can be selected from any desired gene using a CAT (chloramphenicol transferase) vector, a kanamycin vector, or another vector with a selectable marker. Eukaryotic promoters include CMV earliest, HSV thymidine kinase, early and late SV40, retrovirus-derived LTR, and mouse metallothionein-l. Selection of suitable vectors and promoters is well within the skill level of one of ordinary skill in the art and comprises at least one promoter or regulatory promoter operably linked to the nucleic acid encoding the protein or polypeptide according to the present disclosure. Preparations of certain particularly preferred recombinant expression constructs are described herein.

Variants of the polynucleotides of the present disclosure are also contemplated. Variant polynucleotides are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, preferably 95, with one of the polynucleotides of the defined sequence described herein. %, 96%, 97%, 98%, 99% or 99.9% identical, or 0.015M sodium chloride at about 65-68 ° C, 0.0015M sodium citrate or 0.015M chloride at about 42 ° C. It hybridizes to one of the polynucleotides in the defined sequence under stringent hybridization conditions of sodium, 0.0015M sodium citrate and 50% formamide. The polynucleotide variant retains the ability to encode a binding domain or fusion protein thereof having the functionality described herein.

The term "stringent" is used to refer to conditions commonly understood as stringent in the art. Hybridization stringency is primarily determined by temperature, ionic strength, and the concentration of denaturing agents such as formamide. Examples of stringent conditions for hybridization and washing are 0.015M sodium chloride, 0.0015M sodium citrate at about 65-68 ° C, or 0.015M sodium chloride, 0.0015M citrate at about 42 ° C. Sodium and 50% formamide (Sambrook et al. (Et ai), Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 198. Higher stringent conditions (higher temperature, lower ionic strength, higher formamide or other denaturing agents, etc.) can also be used; however, the rate of hybridization will be affected. When hybridization of deoxy oligonucleotides is involved, additional exemplary stringent hybridization conditions are 37 ° C. (for 14-base oligonucleotides), 48 ° C. (for 17-base oligonucleotides), 55 ° C. (for 17-base oligonucleotides). Includes washing with 6 × SSC, 0.05% sodium pyrophosphate at 60 ° C. (for 20 base oligonucleotides) and 60 ° C. (for 23 base oligonucleotides).

A further aspect of the present disclosure is transformed or transfected with any of the polynucleotides or vector / expression constructs of the present disclosure (eg, foristatin polynucleotides or vector / expression constructs) or otherwise containing. , Provide host cells. The polynucleotides or cloning / expression constructs of the present disclosure are any of the methods known in the art, including transformation, transfection and transduction (eg, any of the methods disclosed herein). ) Is introduced into suitable cells. Host cells include, for example, cells of a subject undergoing ex vivo cell therapy, including ex vivo gene therapy. Eukaryotic host cells, which are contemplated as an embodiment of the present disclosure in the presence of the polynucleotides, vectors or proteins according to the present disclosure, are VERO cells in addition to the subject's own cells (eg, human patient's own cells). , HeLa cells, Chinese hamster ovary (CHO) cell line (see US Patent Application Publication No. 2003/0115614, which includes modified CHO cells capable of modifying the glycosylation pattern of the expressed polyvalent binding molecule), COS Cells (COS-7, etc.), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562, HEK293 cells, HepG2 cells, N cells, 3T3 cells, Spodoptera frugiperda cells (eg, Sf9 cells), Saccero Includes cells as well as any other eukaryotic cell known in the art to be useful in the expression and optionally isolation of the proteins or peptides according to the present disclosure. Prokaryotic cells, including Escherichia coli, Bacillus subtilis, Salmonella enterica, Streptomyces, or any prokaryotic cell known in the art to be suitable for expression and optionally isolation of the proteins or peptides according to the present disclosure, are also contemplated. ing. It is contemplated that in the isolation of proteins or peptides from prokaryotic cells, techniques known in the art can be used, in particular for extracting proteins from inclusion bodies. The choice of an appropriate host is within the skill of one of ordinary skill in the art, from the teachings herein. Host cells that glycosylate the fusion proteins of the present disclosure are contemplated.

The term "recombinant host cell" (or simply "host cell") refers to a cell containing a recombinant expression vector. It should be understood that such terms are intended to refer not only to a particular cell of interest, but also to the progeny of such cells. Such progeny may not actually be identical to the parent cell, as certain alterations can occur in progeny due to either mutation or environmental influence, but still the term "host" herein. It is included in the range of "cells". Recombinant host cells can be cultured in conventional nutrient media modified to be suitable for promoter activation, transformant selection or amplification of specific genes. Culture conditions for the particular host cell selected for expression, such as temperature, pH, etc., will be readily apparent to those of skill in the art. Recombinant proteins can also be expressed using a variety of mammalian cell culture systems. As an example of a mammalian expression system, the COS-7 strain of monkey kidney fibroblasts described by Gluzman (1981) Cell Vol. 23, p. 175, as well as other cell lines capable of expressing compatibility vectors, For example, C127, 3T3, CHO, HeLa and BHK cell lines can be mentioned. The mammalian expression vector provides an origin of replication, a suitable promoter and enhancers as needed, as well as any required ribosome binding site, polyadenylation site, splice donor and acceptor site, transcription termination sequence, and 5'adjacent non-transcription. Sequences, such as those described herein in consideration of the preparation of polyadenylation protein expression constructs, will also be included. DNA sequences derived from SV40 splices and polyadenylation sites can be used to provide the required non-transcriptional genetic elements. Introduction of constructs into host cells can be achieved by a variety of methods that may be familiar to those skilled in the art, including calcium phosphate transfection, DEAE-dextran-mediated transfection or electroporation (Davis et al. (1986). Year) Basic Methods in Molecular Biology).

Cells and Compositions In one embodiment, the modified B cells described herein are activated / differentiated in vitro to express a therapeutic agent described herein (eg, follistatin). Transfected as. In one embodiment, the modified B cells described herein are engineered to be activated / differentiated in vitro to express a therapeutic agent described herein (eg, follistatin). (Using a targeted transgene integration approach, such as, for example, zinc finger nuclease, TALEN, meganuclease or CRISPR / CAS9-mediated transgene integration). In one embodiment, the composition has differentiated into trait B cells and has been transfected or otherwise engineered to express one or more proteins of interest (eg, follistatin). Contains B cells. Target cell populations, such as the transfected or otherwise engineered and activated B cell populations of the present disclosure, are pharmaceutical compositions, either alone or in combination with diluents and / or other components such as cytokines or cell populations. It can be administered as a substance.

In one embodiment, modified B cells engineered to express one or more proteins of interest (eg, follistatin) are chemistries specific to the modified B cells after in vitro activation / differentiation. It is collected from the culture at the time when it has the optimum migration ability for the attractant. In some embodiments, optimal migration capacity can be on day 7, 8 or 9 of B cell culture. In some embodiments, optimal migration may be on days 5, 6 or 7 of B cell culture after transfection or manipulation. In some embodiments, optimal migration capacity can be after day 8 of B cell culture after transfection or manipulation, or after day 8 of culture after transfection or manipulation (eg,). On days 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or after day 20). In some embodiments, optimal migration capacity can be prior to day 10 of B cell culture. In some embodiments, optimal migration capacity can be prior to day 8 of B cell culture after transfection or manipulation. In some embodiments, the optimal migration capacity can be on the 6th or 7th day of B cell culture. In some embodiments, optimal migration capacity can be on the 4th or 5th day of B cell culture after transfection or manipulation. In some embodiments, optimal migration capacity can be prior to day 9 of B cell culture. In some embodiments, optimal migration capacity can be prior to day 7 of B cell culture after transfection or manipulation. In some embodiments, optimal migration is optimal for modified B cell homing to CXCL12. In some embodiments, optimal migration is optimal for modified B cell homing into the bone marrow of a subject receiving one or more doses of modified B cells. In some embodiments, B cells are collected for administration to the subject at about 7 to 9 days in culture with optimal migration of CXCL12 and / or the subject to the bone marrow. To. In some embodiments, B cells reach the subject with optimal migration to the bone marrow of CXCL12 and / or the subject at about 5 to about 7 days in culture after transfection or manipulation. Collected for administration. In some embodiments, B cells are collected for administration to the subject prior to about 10 days in culture, with optimal migration of CXCL12 and / or the subject to the bone marrow. In some embodiments, B cells are administered to a subject with optimal migration ability to CXCL12 and / or the subject's bone marrow prior to approximately day 8 in culture after transfection or manipulation. Collected in. In some embodiments, optimal migration is optimal for modified B cell homing to CXCL13. In some embodiments, optimal migration is optimal for modified B cell homing to the site of inflammation in a subject receiving one or more doses of modified B cells. In some embodiments, B cells are collected for administration to the subject at about 6 or about 7 days in culture with optimal migration to the inflamed site in CXCL13 and / or the subject. Will be done. In some embodiments, B cells enter the subject at about 4 or about 5 days in culture after transfection or manipulation, with optimal migration to the inflamed site in CXCL13 and / or the subject. Collected for administration of. In some embodiments, B cells are collected for administration to a subject prior to about 10 days in culture with optimal migration to CXCL13 and / or the site of inflammation. In some embodiments, B cells are collected for administration to a subject prior to about 8 days in culture after transfection or manipulation, with optimal migration to CXCL13 and / or the site of inflammation. Will be done.

In some embodiments, optimal migration is optimal for modified B cell homing to both CXCL12 and CXCL13. In some embodiments, B cells are collected on day 7 of B cell culture with optimal migration capacity for homing to both CXCL12 and CXCL13. In some embodiments, B cells are collected on day 5 of B cell culture after transfection or manipulation, with optimal migration capacity for homing to both CXCL12 and CXCL13.

In some embodiments, engineered B cells are collected when at least about 20% of B cells migrate to a particular chemical attractant in a chemotaxis assay. For example, but not limited to, at least about 20% of B cells produce engineered B cells (eg, FST) as they migrate to CXCL12 in the chemotaxis assay. ) Can be collected. Alternatively, in another non-limiting example, at least about 20% of B cells may collect engineered B cells (eg, produce FST) as they migrate to CXCL13 in a chemotaxis assay. it can. In addition, at least about 30% of B cells migrate to a particular chemoattractant (eg, CXCL12 or CXCL13) in a chemotaxis assay, or at least about 40%, 45%, 50% of B cells. , 55%, 60%, 65% or at least about 70% are engineered B cells (eg, FST) as they migrate to a particular chemoattractant (eg, CXCL12 or CXCL13) in a chemotaxis assay. Can be collected). In addition, more than 70% of B cells can collect engineered B cells (eg, produce FST) as they migrate in the chemotaxis assay. Such chemotaxis assays are known in the art.

Briefly, the cell compositions of the present disclosure are differentiated and activated B cell populations that have been transfected and express the therapeutic agents described herein (eg, follistatin). Can be included in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions include buffers such as neutral buffered saline, phosphate buffered saline, lactated Ringer's solution and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; amino acids such as polypeptides or glycine; Antioxidants; chelating agents such as EDTA or glutathione; adjuvants (eg, aluminum hydroxide); and preservatives can be included. The compositions of the present disclosure are preferably formulated for intravenous or subcutaneous administration.

In one embodiment, the cell composition is evaluated for purity prior to administration. In another embodiment, the cell composition is tested for robustness in therapeutic agent production. In one embodiment, the cell composition is tested for sterility. In another embodiment, the cell composition is screened to confirm compatibility with the recipient subject.

In one embodiment, the engineered B cell population is evaluated for polyclonality prior to administration to the subject. In some embodiments, ensuring polyclonality of the final cell product is an important safety parameter. Specifically, the emergence of predominant clones can be considered as a potential contributor to in vivo tumorigenesis or autoimmune disease. Polychronality can be evaluated by any means known in the art or described herein. For example, in some embodiments, polychronality is assessed by sequencing (eg, deep sequencing) of B cell receptors expressed in the engineered B cell population. Since B cell receptors change during B cell development and become unique among B cells, the method is how many cells share the same B cell receptor sequence (these). Means that is clonal) makes it possible to quantify. Thus, in some embodiments, the greater the number of B cells in an engineered B cell population expressing the same B cell receptor sequence, the higher the chronality of the population, and thus the population's response to administration to a subject. It is less secure. Conversely, in some embodiments, as fewer B cells in an engineered B cell population expressing the same B cell receptor sequence, the population has lower chronality (ie, higher polychronality). Therefore, the safety of the population for administration to the subject is increased.

In some embodiments, the engineered B cells are administered to the subject after it has been determined to be sufficiently polyclonal. For example, engineered B cells can be administered to a subject after it has been determined that a particular B cell clone in the final population does not constitute more than about 0.2% of the total B cell population. Specific B cell clones in the final population account for more than about 0.1% of the total B cell population, or about 0.09%, 0.08%, 0.07%, 0.06%, 0 of the total B cell population. Manipulated B cells can be administered to the subject after it has been determined not to constitute 0.05% or more than about 0.04%. In certain embodiments, engineered B cells (eg, (follistatin)) are used after it has been determined that a particular B cell clone in the final population does not constitute more than about 0.03% of the total B cell population. (Produce) is administered to the subject.

In one embodiment, the cell composition is stored and / or shipped at 4 ° C. In another embodiment, the cell composition is frozen for storage and / or shipping. The cell composition can be frozen, for example, at −20 ° C. or −80 ° C. In one embodiment, the step of freezing the cell composition comprises liquid nitrogen. In one embodiment, the cell composition is frozen using a speed control freezer. Therefore, the methods described herein can further include defrosting steps.

Methods of Use One aspect of the invention relates to in vivo delivery of a therapeutic agent (eg, follistatin) by delivery of modified B cells engineered to express a therapeutic agent (eg, follistatin). In certain embodiments, B cells modified to express follistatin are used in methods of treating and / or preventing chronic diseases and disorders and / or in methods of increasing the size or strength of a patient's muscles. ..

The modified B cells described herein can be administered in a manner appropriate to the disease or disorder to be treated or prevented.

The dose and frequency of administration will be determined by factors such as the patient's condition and the type and severity of the patient's disease, but the appropriate dosage can be determined by clinical trials.

In one embodiment, a single dose of modified B cells is administered to the subject. In one embodiment, two or more doses of modified B cells are administered sequentially to the subject. In one embodiment, three doses of modified B cells are administered sequentially to the subject. In one embodiment, a dose of modified B cells is administered to the subject weekly, biweekly, monthly, bimonthly, quarterly, semi-annually, annually or biennially. In one embodiment, if the amount of therapeutic agent produced by the modified B cells is reduced, a second or subsequent dose of the modified B cells is administered to the subject.

In one embodiment, a dose of modified B cells is present at a certain frequency (eg, weekly, biweekly) until a desired amount (eg, an effective amount) of therapeutic agent (eg, follistatin) is detected in the subject. , Monthly, bimonthly or quarterly) to the subject. In one embodiment, the amount of therapeutic agent (eg, follistatin) is monitored in the subject. In one embodiment, if the amount of therapeutic agent produced by the modified B cells is reduced below the desired amount, subsequent doses of the modified B cells are administered to the subject. In one embodiment, the desired amount is in a range that produces the desired effect. For example, in methods for treating muscular dystrophy (eg, Becker-type muscular dystrophy), the desired amount of follistatin can be the amount in plasma of a subject receiving modified B cells. In some embodiments, the desired amount may be the amount of follistatin that results in a certain level of weight gain by the subject. In some embodiments, the desired amount may be the amount of follistatin that provides a certain level of intensity increase by the subject. In some embodiments, the desired amount may be the amount of follistatin that results in weight gain of a particular level of subject.

When an "effective amount" or "therapeutic amount" is indicated, the exact amount of the composition of the present disclosure to be administered depends on the physician, age, body weight, tumor size, degree of infection or metastasis, and patient ( It can be determined while considering individual differences in the state of the subject). The B cell composition can also be administered multiple times at an appropriate dose (s). Cells can be administered by using injection techniques commonly known in immunotherapy (see, eg, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988).

The optimal dosage and treatment regimen for a particular patient can be determined by one of ordinary skill in the medical arts by monitoring the patient for signs of the disease and adjusting the treatment accordingly. Treatment can also be adjusted after measuring the level of a therapeutic agent (eg, follistatin) in a biological sample (eg, body fluid or tissue sample such as plasma) and can be used to assess treatment efficacy. Treatment can be adjusted accordingly to increase or decrease.

In some aspects of the disclosure, the optimal dose of modified B cells for a multidose regimen first determines the optimal single dose concentration of B cells for the subject, and then the optimal single dose concentration. Decreases the number of B cells present in, resulting in a suboptimal single dose concentration of modified B cells, and this dose of 2 or more of the suboptimal single dose concentration of modified B cells. It can be determined by administration to the subject. In some embodiments, a few or more doses of suboptimal single dose concentrations of modified B cells are administered to the subject. In some embodiments, administration of a single dose concentration of less than optimal single dose concentrations of a few or more doses of modified B cells to a subject is therapeutically engineered to express modified B cells. It results in synergistic in vivo production of the polypeptide. In some embodiments, the suboptimal single dose concentration is 1/2 of the optimal single dose concentration, or 3, 4, 5, 6, 7, 8, 9, 1/10 or 10 minutes. Including less than 1. In some embodiments, the therapeutic polypeptide is follistatin.

In some aspects of the present disclosure, a smaller number of transfected B cells of the present disclosure, in the range of 106 cells / kilogram (106-1011 cells per patient), can be administered. In certain embodiments, the B cells are 1x104, 5x104, 1x105, 5x105, 1x106, 5x106, 1x107, 5x107, 1x108, 5x108. 5, × 109, 1 × 1010, 5 × 1010, 1 × 1011, 5 × 1011 or 1 × 1012 cells are administered to the subject. The B cell composition can be administered multiple times at doses within these ranges. The cells may be autologous or heterologous (eg, allogeneic) to the patient receiving treatment. If desired, the treatment is described herein with mitogen (eg, PHA) or lymphokines, cytokines, and / or chemokines to induce an immune response and enhance engraftment of injected B cells. (For example, GM-CSF, IL-4, IL-6, IL-13, IL-21, Flt3-L, RANTES, MIP1α, BAFF, etc.) can also be included.

Administration of the subject composition can be performed in any convenient manner, including by aerosol inhalation, injection, oral ingestion, infusion, implantation or transplantation. The compositions described herein are subcutaneously, intradermally, intratumorally, intranodally, intramedullarily, intrathecally, intramuscularly (iv). ) Can be administered to the patient by injection or intraperitoneally. The compositions described herein can be administered to a patient directly into the nervous system. In one embodiment, the B cell compositions of the present disclosure are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the B cell compositions described herein are preferably i. v. Administered by injection. The B cell composition can be injected directly into the tumor, lymph node, bone marrow or site of infection.

In yet another embodiment, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump can be used (Langer, 1990, Science 249: 1527-1533; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14: 201; Buchwald et al., 1980; Science 88: 507; Saudek et al., 1989, N. Engl. J. Med. 321: 574). In another embodiment, a polymeric material can be used (Medical Applications of Controlled Release, 1974, Langer and Wise (eds.), CRC Press., Boca Raton, Fla .; Controlled Drug).
Bioavailability, Drug Product Design and Performance, 1984, Smolen and Ball (eds.), Wiley, New York; Ranger and Peppas, 1983; J. Mol. Macromol. Sci. Rev. Macromol. Chem. Vol. 23: see p. 61; Levy et al., 1985, Science Vol. 228: p. 190; During et al., 1989, Ann. Neurol. Volume 25: pp. 351; Howard et al., 1989, J. Mol. Neurosurg. See also Volume 71: Page 105). In yet another embodiment, the controlled release system can be placed in close proximity to the therapeutic target, thereby requiring only a portion of the systemic dose (eg, Medical Applications of Controlled Release, 1984, Ranger). And Wise (eds.), CRC Press., Boca Raton, Fla., Vol. 2, pp. 115-138).

The B cell compositions of the present disclosure can also be administered using any number of matrices. Matrix has been used for many years in the context of tissue engineering (see, eg, Principles of Tissue Engineering (Lanza, Langer and Check), 1997). The present disclosure utilizes such a matrix within the novel context of acting as an artificial lymphoid organ to support and maintain B cells. Therefore, the present disclosure makes use of matrix compositions and formulations that have demonstrated their usefulness in tissue engineering. Thus, the types of matrices that can be used in the compositions, devices and methods of the present disclosure are substantially unlimited and can include both biological and synthetic matrices. In a specific example, U.S. Pat. Nos. 5,980,889; 5,913,998; 5,902,745; 5,843,069; 5,787,900. Alternatively, the compositions and devices described by No. 5,626,561 are utilized. The matrix contains features that are generally associated with biocompatibility when administered to a mammalian host. The matrix can be formed from natural and / or synthetic materials. The matrix can be non-biodegradable; or biodegradable if it is desirable to leave a permanent or removable structure within the animal body, such as an implant. The matrix can take the form of sponges, implants, tubes, telfa pads, fibers, hollow fibers, lyophilized components, gels, powders, porous compositions or nanoparticles. In addition, the matrix can be designed to allow sustained release seeded cells or producing cytokines or other active agents. In certain embodiments, the matrix of the present disclosure is a semi-solid that is flexible and elastic and permeable to substances such as inorganic salts, aqueous fluids, and dissolved gaseous agents containing oxygen. It can be described as a scaffold.

The matrix is used herein as an example of a biocompatible material. However, the present disclosure is not limited to matrices, and thus whenever the term matrix (s) appears, such terms allow cell retention or crossing of cells and are biocompatible. Includes devices and other substances that can be used directly through this substance or in combination with certain semipermeable substances to allow crossing of macromolecules, such that the substance itself is a semipermeable membrane. It should be deciphered as a thing.

In certain embodiments of the present disclosure, B cells transfected and activated using the methods described herein or other methods known in the art are agents such as antiviral agents. , Chemotherapy, irradiation, immunosuppressants such as cyclosporine, bisulfin, bortezomib, azathiopurine, methotrexate, mycophenolate and FK506, antibodies, or other immunosuppression such as campus, anti-CD3 antibody or other antibody therapy Any number of agents (immunoablative agent), cytoxin, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and treatments with, but not limited to, irradiation. It is administered to the patient in conjunction with the relevant treatment modality (eg, before, at the same time, or afterwards). These drugs inhibit either the calcium-dependent phosphatase calcineurin (cyclosporine and FK506), the proteasome (bortezomib), or the p70S6 kinase, which is important for growth factor-induced signaling (rapamycin) (liu et al., Cell). 66: 807-815, 1991; Henderson et al., Immuno. 73: 316-321, 1991; Bierer et al., Curr. Opin. Immuno. 5: 763-773, 1993; Isoniemi (above). reference)). In a further embodiment, the cell compositions of the present disclosure are T cells using either bone marrow transplantation, fludarabine, external irradiation therapy (XRT), chemotherapeutic agents such as cyclophosphamide, or antibodies such as OKT3 or Campus. It is administered to the patient in conjunction with ablation therapy (eg, before, at the same time, or after). In one embodiment, the cell compositions of the present disclosure are administered after B cell depletion therapy, such as an agent that reacts with CD20, such as Rituxan®. In one embodiment, the cell composition of the present disclosure is administered after B cell depletion therapy using a drug such as bortezomib. For example, in one embodiment, the subject can receive a peripheral blood stem cell transplant following standard treatment with high-dose chemotherapy. In certain embodiments, after transplantation, the subject receives an injection of expanded immune cells of the present disclosure. In additional embodiments, the expanded cells are administered before or after surgery.

The dose of the above-mentioned treatment to be administered to the patient will vary with the condition being treated and the exact nature of the recipient of the treatment. Dosage scaling for human administration can be performed according to practices accepted in the art.

Modified B cells can be used in the treatment or prevention of various diseases and disorders. In certain embodiments, B cells modified to express follistatin are used in a method of increasing the size or strength of a patient's muscle. For example, the present disclosure provides a method of increasing the muscle size or strength of a subject, comprising the step of administering an effective amount of B cells expressing a follistatin polypeptide. Increases in muscle size or strength can generally occur throughout the subject or in targeted muscles. Muscular dystrophy (eg, Duchenne muscular dystrophy, Becker muscular dystrophy, Emeridorefus muscular dystrophy, limb dystrophy, muscular dystrophy syndrome, Ulrich syndrome, Fukuyama muscular dystrophy, Walkerwarburg syndrome, muscle-eye-brain disease, facial muscular dystrophy Muscular dystrophy), congenital muscular dystrophy, muscular dystrophy (Steinart's disease), non-dystrophy myotony, muscular dystrophy spinal muscular dystrophy, familial muscular dystrophy lateral sctrophy, hereditary muscular dystrophy, scharcomery Tooth disease, chronic inflammatory dystrophy, distal muscular dystrophy, muscular dystrophy / central muscular dystrophy, nemarin dystrophy, minicore disease, central core disease, desminopathy, inclusion myitis, muscular dystrophy, congenital muscular dystrophy syndrome, muscular dystrophy Disorders, as well as the disorders described in Emery (2002) The Lancet, 359: 687-695 and Khurana et al. (2003) Nat. Rev. Drug Disc., 2: 379-386, inflammatory muscular disorders (eg,). Muscular dystrophy), muscular dystrophy or trauma, muscular dystrophy (which can occur after long-term muscular rest or limb fixation) and muscular atrophy or weakness as a result of aging, cancer or various types of chronic illnesses. Targeted muscles can be damaged, muscularly weakened, or deficient, as is possible in the case of various muscular disorders, including. For example, in some cases, muscle can be damaged, weakened, or deficient due to muscle weakness. In some cases, muscle can be damaged, weakened, or lost due to spinal muscular atrophy (SMA). In some cases, muscle can be damaged, weakened, or defective by amyotrophic lateral sclerosis (ALS). In some cases, muscles can be damaged, weakened, or lost due to Pompe disease. The method can also increase muscle size or strength in healthy muscles.

In some embodiments, the disclosure is a method for treating a disease or disorder in an individual expressing a B cell genetically modified to express follistatin (eg, a follistatin + FST344 transcript variant). B cells) to be administered to a subject in need thereof.

In some embodiments, the disclosure is a method for treating muscular disorders in an individual, expressing B cells genetically modified to express follistatin (eg, follistatin + FST344 transcript variant). B cells) include the step of administering to a subject having or suspected of having such a muscular disorder, providing a method in which the muscular disorder is muscular dystrophy. In some embodiments, the muscular dystrophy is Dusenne's muscular dystrophy, Becker's muscular dystrophy, Emeridorefus's muscular dystrophy, muscular dystrophy, muscular dystrophy, Ulrich syndrome, Fukuyama muscular dystrophy, Walkerwarburg syndrome, muscular-eye-brain disease. , Facial dystrophy, muscular dystrophy, congenital muscular dystrophy, muscular dystrophy (Steinart's disease), non-dystrophy muscular dystrophy, periodic muscular dystrophy, muscular dystrophy, familial muscular dystrophy, hereditary muscular dystrophy Muscular dystrophy, strophy dystrophy, chronic inflammatory dystrophy, distal muscular dystrophy, muscular dystrophy / muscular dystrophy, nemarin dystrophy, minicore disease, muscular dystrophy, desminopathy, muscular dystrophy, micular dystrophy, muscular dystrophy congenital muscular dystrophy , Post-porio muscular dystrophy, and selected from the disorders described in Emery (2002) The Lancet, 359: 687-695 and Khurana et al. (2003) Nat. Rev. Drug Disc., 2: 379-386. To.

In some embodiments, the disclosure is a method for treating muscle disorders in an individual, expressing B cells genetically modified to express follistatin (eg, follistatin + FST344 transcript variant). B cells) include the step of administering to a subject having or suspected of having such a muscle disorder, providing a method in which the muscle disorder is an inflammatory muscle disorder. In some embodiments, the inflammatory myopathy is inclusion body myositis.

In some embodiments, the disclosure is a method for treating muscle disorders in an individual, expressing B cells genetically modified to express follistatin (eg, follistatin + FST344 transcript variant). B cells) include the step of administering to a subject having or suspected of having such a muscle disorder, providing a method in which the muscle disorder is muscle injury or trauma.

In some embodiments, the disclosure is a method for treating muscle disorders in an individual, expressing B cells genetically modified to express follistatin (eg, follistatin + FST344 transcript variant). B cells), including the step of administering to a subject having or suspected of having such muscle damage, in muscle disuse (eg, after prolonged floor rest or limb fixation). Provide a method.

In some embodiments, the disclosure is a method for treating muscle weakness in an individual, expressing B cells genetically modified to express follistatin (eg, follistatin + FST344 transcript variant). B cells), including the step of administering to a subject having or suspected of having such muscle damage, muscle atrophy or muscle atrophy as a result of age, cancer or various types of chronic disease. Provide a method of choice from muscle weakness.

In some embodiments, the present disclosure is a method for treating an individual exhibiting mild, moderate or severe muscle weakness, muscle weakness, and / or effects on independent gait and expresses follistatin. Provided is a method comprising the step of administering to a subject a B cell thus genetically modified (eg, a B cell expressing a follistatin + FST344 transcript variant).

In some embodiments, the present disclosure describes mild, moderate or severe muscle fragility, muscle hypertrophy, muscular hypertrophy, arthrogryposis, skeletal deformity, myopathy, swallowing disorders, intestinal and bladder dysfunction, A method for treating individuals with muscular ischemia, cognitive deficits, behavioral dysfunction, social dysfunction, spinal kyphosis, and / or respiratory dysfunction, genetically modified to express follistatin. Provided is a method comprising the step of administering to a subject B cells (eg, B cells expressing a foristatin + FST344 transcript variant).

In certain embodiments, the present disclosure is a method for treating muscular dystrophy in an individual and is a B cell that is genetically modified to express follistatin (eg, a B cell that expresses a follistatin + FST344 transcript variant). ) Is administered to a subject having or suspected of having Becker-type muscular dystrophy.

In some embodiments, a single maximum effective dose of follistatin + B cells (eg, B cells expressing the follistatin + FST344 transcript variant) is administered to the subject. In some embodiments, two or more doses of follistatin + B cells (eg, B cells expressing the follistatin + FST344 transcript variant) are administered to the subject, thereby engrafting the follistatin. Maximize the amount of + B cells. In some embodiments, a dose of 2 or more doses of follistatin + B cells administered to the subject (eg, B cells expressing the follistatin + FST344 transcript variant) is a single maximum effective dose of follistin. Contains less follistatin + B cells than statin + B cells. In some embodiments, 2 or more doses of follistatin + B cells (eg, B cells expressing the follistatin + FST344 transcript variant) are below the maximum effective single dose of follistatin + B cells. When administered to a subject at a dose of + B cells, a resulting synergistic increase in follistatin production occurs. In one embodiment, the step of administering follistatin + B cells to a subject results in the normal levels of follistatin found in a healthy control subject. In one embodiment, the step of administering follistatin + B cells to a subject results in follistatin above normal levels in the subject. In one embodiment, the step of administering follistatin + B cells (eg, B cells expressing the follistatin + FST344 transcript variant) to a subject increases the intensity of the subject. In one embodiment, the step of administering follistatin + B cells (eg, B cells expressing the follistatin + FST344 transcript variant) to the subject increases the intensity of the subject as compared to normal levels. In one embodiment, the step of administering to a subject follistatin + B cells (eg, B cells expressing the follistatin + FST344 transcript variant) prevents loss of subject strength.

(Example 1)
Generation of B cells expressing follistatin Sleeping beauty transposons and transposase constructs for transposition and expression of human follistatin (FST) were generated. Transposons were assembled to achieve FST gene integration and expression in B cells. We used the EEK promoter, which consists of a promoter and enhancer element derived from the human immunoglobulin gene, as well as other regulatory elements previously described, to achieve high levels of expression in B cells. To test for FST translocation and expression, human B cells were isolated from two separate donors, expanded in culture in B cell culture medium, incubated in a 37 ° C. incubator with 5% CO2, and 3 On day, mRNA encoding pKT2 / EEK-FST344 plus SB100x transposase was electroporated. Cell lysates prepared 2, 5, 8, and 11 days after electroporation (ie, days 5, 7, 11, and 14 of culture) were significantly more than wild-type untransfected cells. It contained increased FST, demonstrating the effectiveness of the SB transposon system in achieving high levels of FST expression in expanded and proliferated human B cells (FIG. 5A). Notably, FST expression was slightly reduced from day 5 (possibly due to early episome FST expression) and persisted from day 7-14, which is to the B cells of the construct. It suggested stable incorporation. In fact, RT-PCR confirmed the stable integration of FST insertion into the B cell genome (Fig. 5B). Such FST-expressing B cells are referred to as FST + B cells in the following examples.

(Example 2)
In vivo Production of FST In order to determine whether B cells engineered according to the present disclosure can promote an in vivo increase in FST production, we have generated FST + B cells in Example 1. Was injected into wild-type mice.

^{Specifically, 4 mice had 2 × 10 6} human FST + B cells diluted in vehicle (500 μl phosphate buffered saline (PBS)) or vehicle (PBS) to 500 μl on day 0. Received an intravenous (tail vein) injection. ^{In addition, on day -7, 3 × 10 6} primary autologous blood cells enriched with respect to CD4 + T cells were injected intraperitoneally (i.p.) into mice to pKT2 / EEK-FST Plus SB100x transposer. Provided support for mRNA transposable B cells encoding ze. We observed a 2-fold increase in human FST in FST + B cell-treated mouse plasma, demonstrating strong evidence of successful human B cell adoption (Fig. 1). FST levels peaked approximately 28 days after injection and dropped to near normal levels by 35 days. Although not performed in FST-deficient animals, the results of this experiment nevertheless provide an example of human FST levels that can be achieved after introduction of a highly potent FST + B cell population into wt mice. We found that plasma levels of FST correlate with plasma levels of human IgG, thus providing evidence of adoption and engraftment of FST + B cells (FIGS. 2A-2D).

To determine if FST + B cells had any effect on treated mice, we monitored the body weight of control mice and mice treated with FST + B cells 35 days after injection. As shown in FIG. 3, FST + B cells grew on average 4.1% (0.9 grams) more than mice treated with vehicle controls. In addition, weight gain was treated with forelimb grip test (16% improvement in mice treated with FST + B cells compared to vehicle control) (FIG. 4A); limb grip test (treated with FST + B cells compared to vehicle control). 23% improvement in mice) (FIG. 4B); and a significant intensity improvement in a hanging test (23% improvement in mice treated with FST + B cells compared to vehicle control) (FIG. 4C).

Thus, such data demonstrate that B cells can be used in the methods disclosed herein to express FST and deliver it to a subject, inducing weight gain and improved strength. To.

Further embodiments can be provided by combining the various embodiments described above. All US patents, US patent application publications, US patent applications, foreign patents, foreign patent applications and non-patent publications referenced herein and / or listed in the application datasheet are in their entirety. To be incorporated herein by. The embodiments may be modified if it is necessary to use the concepts of various patents, applications and publications to provide yet another embodiment.

Based on the above detailed description, the above and other modifications can be made to the embodiments. In general, the terms used in the following claims should not be construed as limiting the scope of the claims to the specific embodiments disclosed in the specification and claims. , Such claims should be construed to include all possible embodiments, along with the full scope of equality to which they are entitled. Therefore, the scope of claims is not limited by this disclosure.

Claims (85)

Recombinant B cells containing the follistatin gene. The B cell according to claim 1, wherein the follistatin gene is operably linked to a promoter. The B cell according to claim 1 or 2, wherein the follistatin gene is a human follistatin gene. The B cell according to any one of claims 1 to 3, wherein the follistatin gene is a splice site variant of human follistatin FST-344. The B cell according to any one of the preceding claims, wherein the B cell is a human B cell. The B cell according to any one of the preceding claims, wherein the B cell has been transduced or translocated with the follistatin gene. The B cell according to any one of claims 1 to 6, wherein the B cell contains the follistatin gene because the follistatin gene has been transduced using a transposon system. The B cell according to claim 7, wherein the transposon system is a sleeping beauty transposon system or a Piggybac transposon system. The B cell according to any one of claims 1 to 7, wherein the B cell expresses the follistatin gene by transduction with a virus carrying the follistatin gene. The method according to any one of claims 1 to 7, wherein the B cell is transduced by a retrovirus containing the follistatin gene, a lentivirus, an adenovirus, or an adeno-associated virus, and thus contains the follistatin gene. B cells. The B cell according to any one of claims 1 to 7, wherein the B cell is engineered to contain the follistatin gene using a targeted integration technique. A claim that said targeting integration utilizes one or more zinc finger nucleases, transcriptional activator-like effector nucleases (TALENs), and / or CRISPR / Cas9 systems, including but not limited to CRISPR / Cas9 systems. 11. The B cell according to 11. Nucleic acid encoding follistatin using a method in which the B cells are selected from the group consisting of retroviral vectors, lentiviral vectors, adeno-associated virus vectors, adenoviral vectors, any other RNA or DNA viral vectors. By introducing the follistatin gene into a non-viral DNA encoding a follistatin that has been introduced using chemical or physical means such as lipofection, polycation complex formation, electroporation, etc. The B cell according to any one of claims 1 to 7, which is engineered to contain RNA. The B cell according to any one of the preceding claims, wherein the follistatin protein is secreted by the recombinant B cell. A method of delivering follistatin to a subject, comprising the step of administering recombinant B cells containing the follistatin gene. A method of delivering follistatin to a subject in need thereof, comprising the step of administering the recombinant B cells according to any one of claims 1-14. The method of claim 15 or 16, wherein the subject is a mammal. The method according to any one of claims 15 to 17, wherein the subject is a human. The method of any one of claims 15-18, wherein the subject has muscular dystrophy. The method according to any one of claims 15 to 19, wherein the subject has Becker-type muscular dystrophy. The method of any one of claims 15-20, wherein the step of administering the recombinant B cells to the subject results in treatment of the subject's disease, disorder, or condition. The method of any one of claims 15-20, wherein the step of administering the recombinant B cells to the subject results in treatment of muscular dystrophy. The method according to any one of claims 15 to 22, wherein the step of administering the recombinant B cells to the subject increases the body weight of the subject. 23. The method of claim 23, wherein the subject gains at least about 4% body weight. 24. The method of claim 24, wherein significant weight gain occurs within 30 days. 24. The method of claim 24, wherein significant weight gain occurs in about 30 days. The method according to any one of claims 15 to 26, wherein the step of administering the recombinant B cells to the subject increases the muscle mass of the subject. The method according to any one of claims 15 to 27, wherein the step of administering the recombinant B cells to the subject increases the strength of the subject. The method of any one of claims 15-28, wherein the administration step of the recombinant B cells raises plasma levels of follistatin in the subject. A method of treating, preventing, or alleviating myopathy by administering recombinant B cells containing the follistatin gene. A method for treating, preventing, or alleviating muscular dystrophy by administering the recombinant B cells according to any one of claims 1 to 13. The recombinant B according to any one of claims 1 to 13, wherein the recombinant B cell is derived from a B cell obtained from the subject or a B cell derived from a cell obtained from the subject. cell. The recombinant B cell according to claim 32, wherein the recombinant B cell is derived from a B cell progenitor cell obtained from the subject. The recombinant B cell according to claim 32, which is derived from a cell obtained from the subject, in which the recombinant B cell is differentiated into the B cell or a progenitor cell of the B cell. The recombinant B cells
(A) With the step of collecting and isolating immune cells from the blood of the subject;
(B) The step of transducing the DNA encoding the follistatin into the cells;
(C) With the step of expanding and proliferating the selected cells ex vivo;
(D) The set according to any one of claims 1 to 13 and 32 to 34, which is manipulated by a step of differentiating the expanded and proliferated cells into plasma cells and / or plasma blasts by ex vivo. Alternate B cells. The recombinant B cell according to claim 35, wherein the immune cell isolated from step a is a CD19-positive cell. The recombinant B cell according to claim 35 or 36, wherein the transduction step of step b is electroporation. The recombinant B cell according to claim 37, wherein the sleeping beauty transposon system is used for the electroporation. The recombinant B cell according to any one of claims 35 to 38, wherein the differentiated cells are CD38 (+) and CD20 (−). A method comprising the step of administering to a subject the recombinant B cells according to any one of claims 35 to 39. The method of any one of claims 15-31 and 35-40, comprising the step of administering to the subject two or more sequential doses of genetically modified B cells. 41. The method of claim 41, wherein the step of administration comprises 2 or more doses of the genetically modified B cells at a single dose concentration below optimal. 41. The method of claim 41, wherein the step of administration comprises 3 or more doses of genetically modified B cells. 41. The method of claim 41, wherein the genetically modified B cells are autologous to the subject. 41. The method of claim 41, wherein the genetically modified B cells are allogeneic to the subject. 41. The method of claim 41, wherein the subject is a human. 41. The method of claim 41, wherein the genetically modified B cells are CD20-, CD38- and CD138-. 41. The method of claim 41, wherein the genetically modified B cells are CD20-, CD38 + and CD138 +. 41. The method of claim 41, wherein the genetically modified B cells are CD20-, CD38 + and CD138-. 41. The method of claim 41, wherein the administering step comprises intravenous, intraperitoneal, subcutaneous, intrathecal, intrathecal or intramuscular injection. The method of claim 50, wherein the administering step comprises an intravenous injection. The method according to any one of claims 15 to 31 and 35 to 51, wherein the genetically modified B cells are engineered on the second or third day after culturing. 52. The method of claim 52, wherein the genetically modified B cells are engineered using a method comprising electroporation. (A) The genetically modified B cells were collected for administration to a subject on days ranging from days 1 to 12 of in vitro culture.
(B) The genetically modified B cells are collected for administration to a subject on day 4, 5, 6, or 7, or 8 of the post-manipulation culture.
The method according to any one of claims 15 to 31 and 35 to 53. According to any one of claims 15 to 31 and 35 to 54, wherein the genetically modified B cells are collected for administration to a subject on or after 8 days from the start of post-procedure culture. The method described. 55. The method of claim 55, wherein the genetically modified B cells are collected for administration to a subject 10 days or earlier from the start of culturing after manipulation. The method of any one of claims 15-31 and 35-56, wherein the collected genetically modified B cells do not produce significant levels of inflammatory cytokines. The method of any one of claims 15-31 and 35-57, which is collected at a time in culture where it is determined that the genetically modified B cells do not produce significant levels of inflammatory cytokines. The genetically modified B cells were placed in a culture system containing IL-2, IL-4, IL-10, IL-15, IL-31 and multimerized CD40 ligand, respectively, throughout the pre- and post-manipulation culture period. The method of any one of claims 15-31 and 35-58 for growing. 59. The method of claim 59, wherein the multimerized CD40 ligand is a HIS-tagged CD40 ligand that is multimerized using an anti-his antibody. The method according to any one of claims 15 to 31 and 35 to 60, further comprising the step of expanding and proliferating the genetically modified B cells prior to the step of administering to the subject. The method of claim 61, wherein the final population of expanded and proliferated genetically modified B cells exhibits a high degree of polyclonality. 61. The method of claim 61, wherein any particular B cell clone in the final population of expanded and proliferated genetically modified B cells constitutes less than 0.2% of the total B cell population. 16. The method of claim 61, wherein any particular B cell clone in the final population of expanded and proliferated genetically modified B cells constitutes less than 0.05% of the total B cell population. The method of any one of claims 15-31 and 35-64, wherein the genetically modified B cell comprises a polynucleotide encoding a selectable marker. 65. The method of claim 65, wherein the selectable marker is a human DHFR gene with enhanced resistance to methotrexate. The method of claim 66, wherein the human DHFR gene with enhanced resistance to methotrexate comprises a leucine-to-tyrosine substitution mutation at amino acid 22 and a phenylalanine-to-serine substitution mutation at amino acid 31. The method of any one of claims 15-31 and 35-67, comprising treating the genetically modified B cells with methotrexate prior to collection for administration. The method of claim 68, wherein the treatment with methotrexate is between 100 nM and 300 nM. The method of claim 69, wherein the treatment with methotrexate is 200 nM. The method according to any one of claims 15 to 31 and 35 to 70, wherein when the genetically modified B cell is administered to the subject, it migrates to various tissues. At least one of the population of genetically modified B cells administered to the subject is selected from the group consisting of bone marrow, intestine, muscle, spleen, kidney, heart, liver, lung and brain1 The method of any one of claims 15-31 and 35-71, which migrates to a species or tissue. At least one of the population of genetically modified B cells administered to the subject migrates to the subject's bone marrow, intestines, muscles, spleen, kidneys, heart, liver, lungs and brain. , The method of claim 72. Modified B cells transduced to express the follistatin gene and the DHFR gene. A method for treating a myopathy that comprises administering to a subject B cells genetically modified to express follistatin. The method of claim 75, wherein the muscle disorder is selected from muscular dystrophy, inflammatory muscle disorder, muscle injury or trauma, muscle disuse, and muscle atrophy or weakness. The method of claim 75 or 76, wherein the muscular dystrophy is Duchenne muscular dystrophy, Becker muscular dystrophy or facial scapulohumeral muscular dystrophy. The method of claim 75 or 76, wherein the inflammatory myopathy is inclusion body myositis. The method of claim 75 or 76, wherein the muscle disuse occurs after prolonged bed rest or limb fixation. The method of claim 75 or 76, wherein the muscle atrophy or weakness is caused by aging, cancer or chronic disease. The method of claim 75, wherein the muscle disorder is muscle hypoplasia. The method of claim 75, wherein the muscular disorder is spinal muscular atrophy (SMA). The method of claim 75, wherein the muscular disorder is amyotrophic lateral sclerosis (ALS). The method of claim 75, wherein the muscle disorder is Pompe's disease. The method of any one of claims 75-84, wherein the follistatin comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-4.

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